Bifunctional molecules and therapies based thereon

ABSTRACT

Bifunctional molecules and methods for their use in the production of binary complexes in a host are provided. The bifunctional molecule is a conjugate of a drug moiety and a presenter protein ligand. In the subject methods, an effective amount of the bifunctional molecule is administered to the host. The bifunctional molecule binds to the presenter protein to produce a binary complex that exhibits at least one of improved affinity, specificity or selectivity as compared to the corresponding free drug. The subject methods and compositions find use in a variety of therapeutic applications.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Divisional of U.S. patent application Ser.No. 09/316,932, filed May 21, 1999 which claims priority to the filingdate of the U.S. Provisional Patent Application Serial No. 60/086,451filed May 22, 1998, the disclosure of which is herein incorporated byreference.

ACKNOWLEDGMENT

[0002] This invention was made with United States Government supportunder Grant No. CA39612 awarded by National Institutes of Health. TheUnited States Government has certain rights in this invention.

INTRODUCTION

[0003] 1. Technical Field

[0004] The field of this invention is pharmacology.

[0005] 2. Background of the Invention

[0006] Any chemical agent that affects any process of living is a drug.Drugs are a critical tool for health care practitioners, as they areused in the prevention, diagnosis and treatment of disease. Because oftheir criticality to the health care profession, annual world investmentinto the research and development of new chemical agents withtherapeutic potential reaches into the billions of dollars. As a result,a large number of drugs have been developed to date and new chemicalagents having potential therapeutic utility are frequently discovered.Chemical agents that find, or have found, use as drugs include naturallyoccurring and synthetic small molecules, as well as larger molecules,such as proteinaceous compounds.

[0007] Most small molecule drugs cause a pharmacological effect bybinding to a target protein and altering the pharmacological activity ofthe target in some way. For a given small molecule drug, it is desirablethat the drug have at least one of high affinity and specificity for itstarget. If a small molecule has high affinity for its target, it ischaracterized by having good binding to its target. If a small moleculehas specificity for its target, it is characterized by havingdifferential affinity between its target and other, non-target proteins.Besides displaying high affinity and/or specificity, a given smallmolecule should be selective with respect to the cell or tissue in whichit affects a biological activity. Selectivity will assure that the drugtarget will be affected by the drug only in cells involved in thedisease process.

[0008] Screens for small molecule drugs rarely identify high affinityligands, low affinity ligands with high specificity or selectiveligands. Much more often, compounds are identified that have biologicalactivity but with relatively low affinity and low specificity for theirtargets. Furthermore, identified compounds usually lack selectivity fortheir targets with respect to cell or tissue type. Because of this lowaffinity, specificity, or selectivity or combination thereof, theseidentified ligands never find clinical use.

[0009] As such, of great interest to the pharmaceutical industry andrelated fields would be the development of a method for increasing atleast one of the affinity, specificity and selectivity of thesepreviously identified biologically active agents, such that agents thatotherwise lack sufficient affinity and/or specificity nonetheless couldfind clinical utility.

[0010] Relevant Literature

[0011] Patent publications of interest include: WO 91/01743; WO94/18317; WO 95/02684; WO 95/10302; WO 96/06111; WO 96/12796; WO96/13613; WO 97/25074; WO 97/29372; WO 98/11437; WO 98/47916; U.S. Pat.Nos. 5,830,462; 5,843,440; and 5,871,753. References of interestinclude: Briesewitz et al., Proc. Nat'l Acad. Sci. USA (March 1999) 96:1953-1958; Clardy, Proc. Nat'l Acad. Sci. USA (March 1999) 1826-1827;Crabtree & Schreiber, Elsevier Trends Journal (November 1996) 418-422;Spencer et al., Curr. Biol. (July 1996) 6:839-847; Spencer et al.,Science (1993) 262: 1019; Chakraborty et al., Chem. & Biol. (March 1995)2:157-161; Ho et al., Nature (1996) 382: 822; Riviera et al., NatureMedicine (1996) 2: 1028; Klemm et al., Current Biology (1997) 7: 638;Belshaw et al., Proc. Nat'l. Acad. Sci. USA (1996) 93: 4604; Livnah etal., Science (1996) 273: 464; Johnson et al., Chemistry and Biology,(1997) 4: 939; Garboczi et al., Nature (1996) 384:134; Kissenger et al.,Nature (1995) 378:641; Griffith et al., Cell (1995) 82: 507; Choi etal., Science (1996) 273:239. Also of interest are Kramer et al., J.Biol. Chem. (1992) 267:18598-18604; and Varshavsky, Proc. Nat'l Acad.Sci. USA (March 1998) 95: 2094-2099; Varshavsky, Proc. Nat'l Acad. Sci.USA (April 1995) 92:3663-3667; and Mu et al., Biochem. Biophys. Res.Comm. (1999)255:75-79.

SUMMARY OF THE INVENTION

[0012] Bifunctional molecules capable of producing at least a binarycomplex with a host endogenous presenter protein are provided. In thesubject methods, a bifunctional molecule is synthesized by covalentlylinking an endogenous presenter protein ligand to a drug moiety, eitherdirectly or through a linking group. An effective amount of abifunctional molecule is administered to the host, where thebifunctional molecule binds to the endogenous presenter protein toproduce the binary complex. The resultant binary complex modulates (e.g.enlarges) the target binding surface area of the drug moiety, i.e. thesurface area available for binding with the target, in a manner suchthat at least one of enhanced affinity, specificity or selectivity areobserved as compared to that observed with the free drug.

[0013] In one embodiment where the binary complex results in enhancedaffinity, a tripartite complex is produced in which binding interactionsare present between the presenter and target as well as the drug moietyand target. In a second embodiment where the binary complex results inenhanced specificity, delivery of the drug as a bifunctional moleculeresults in differential affinity with respect to its potential targetssuch that the drug moiety binds more to its desirable target than to itsundesirable targets as compared to a free drug control situation, e.g.where tripartite complexes produced between the binary complex andundesired targets are characterized by the presence of unfavorableinteractions while tripartite complexes produced between the binarycomplex and the desired target are characterized by the presence of nointeractions, neutral interactions or favorable interactions. In a thirdembodiment where the binary complex results in enhanced selectivity,tripartite complexes produced with undesired targets in a first cell ortissue type are characterized by the presence of unfavorableinteractions while the bifunctional molecule affects the desired targetin a second cell or tissue type, e.g. a second type of cell or tissue inthe same organism or a microorganism in a host organism, withoutformation of a binary complex between the bifunctional molecule and apresenter protein due to the lack of the presenter protein.

[0014] The subject methods and compositions find use in a variety oftherapeutic applications.

BRIEF DESCRIPTION OF THE FIGURES

[0015]FIG. 1 provides an illustration of the general concept of thesubject invention.

[0016]FIG. 2 provides the structures of FKpYEEI and SLFpYEEI.

[0017]FIG. 3 provides a representation of a competition binding assay ofFyn-SH2 domain to pYEEI beads in the presence of FKpYEEI and FKpYEEIplus FKBP52.

[0018]FIG. 4 shows the results from a competition binding assay of theFyn-SH2 domain to pYEEI beads in the presence of FKpYEEI and FKpYEEIplus FKBP52.

[0019]FIG. 5 shows the results from a competition binding assay of theFyn SH2 domain to pYEEI beads in the presence of FKpYEEI, FKpYEEI plusFKBP52 and FKpYEEI plus FKBP52 plus FK506.

[0020]FIG. 6 shows the results from a competition binding assay of theFyn SH2 domain to pYEEI beads at various concentrations of pYEEI andFKpYEEI in the presence and absence of FKBP52.

[0021]FIG. 7 shows the results from a competition binding assay of theFyn SH2 domain to pYEEI beads in the presence of FKpYEEI and FKpYEEIplus FKBP12.

[0022]FIG. 8 shows the results from a competition binding assay of thePLC SH2 domain to pYEEI beads in the presence of FKpYEEI and FKpYEEIplus FKBP12.

[0023]FIG. 9 shows the results from a competition binding assay of thePLC SH2 domain to pYEEI beads in the presence of FKpYEEI, FKpYEEI plusFKBP12 and FKpYEEI plus FKBP12 plus FK506.

[0024]FIG. 10 shows the results from a competition binding assay of theFyn, Lck and PLC SH2 domains to pYEEI beads in the presence of FKpYEEI,FKpYEEI plus FKBP12 and FKpYEEI plus FKBP12 plus FK506.

[0025]FIG. 11 shows the results from a competition binding assay of theFyn SH2 domain in the presence of SLFpYEEI and SLFpYEEI plus FKBP12.

[0026]FIG. 12 shows the results from a competition binding assay of theFyn SH2 domain to pYEEI beads in the presence of SLFpYEEI, SLFpYEEI plusFKBP12 and SLFpYEEI plus FKBP12 plus FK506.

[0027]FIG. 13 shows the results from a competition binding assay of theFyn SH2 domain to pYEEI beads at increasing concentrations of SLFpYEEIin the presence and absence ofFKBP12.

[0028]FIG. 14 shows the results from a competition binding assay of theFyn, Lck and PLC SH2 domains in the presence of SLFpYEEI, SLFpYEEI plusFKBP 12 and SLFpYEEI plus FKBP12 plus FK506.

DEFINITIONS

[0029] The term “affinity” refers to the nature of the binding of afirst molecule to a second molecule, e.g. a drug moiety to its target, apresenter protein ligand to its presenter protein, etc. In other words,affinity is used to describe how strong a first molecule binds to asecond molecule. The affinity of a bifunctional molecule of the subjectinvention to its target and its presenter protein can readily bedetermined by competitive binding assays or thermodynamic methods, asdescribed in the experimental section infra.

[0030] The term “bifunctional molecule” refers to a non-naturallyoccurring molecule that includes a presenter protein ligand and a drugmoiety, where these two components may be covalently bonded to eachother either directly or through a linking group.

[0031] The term “binary complex” refers to a complex that is made up ofa bifunctional molecule and its corresponding presenter protein, i.e.the complex that results from binding of the presenter protein ligand ofthe bifunctional molecule to the presenter protein.

[0032] The term “drug” refers to any active agent that affects anybiological process.

[0033] Active agents which are considered drugs for purposes of thisapplication are agents that exhibit a pharmacological activity. Examplesof drugs include active agents that are used in the prevention,diagnosis, alleviation, treatment or cure of a disease condition.

[0034] By “pharmacologic activity” is meant an activity that modulatesor alters a biological process so as to result in a phenotypic change,e.g. cell death, cell proliferation etc. The term “specificity” is usedto describe the affinity profile of a drug with respect to its potentialtargets, i.e. it characterizes the differential affinity of a drug forits potential targets. The specificity of a drug can readily beascertained by determining the affinity of a drug for each of itspotential targets (i.e. how well the drug binds to each of its potentialtargets) and comparing the observed affinities to obtain an affinityprofile of the drug. The term “selectivity” is used to characterize theactivity profile of an active agent with respect to two or moredifferent cell types. In other words, selectivity collectively describesthe comparative activity of a drug in two or more different types ofcells.

[0035] The term “tripartite complex” refers to a complex that is made upof a drug target, a bifunctional molecule and a presenter protein.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0036] Bifunctional molecules, synthesis and screening methods, andmethods for their use in the production of at least binary complexes ina host are provided. The bifunctional molecules have a drug moietycovalently linked to a presenter protein ligand, either directly orthrough a linking group. In the subject methods, an effective amount ofthe bifunctional molecule is administered to the host. Uponadministration, the bifunctional molecule binds to the presenter proteinto produce the binary complex. The binary complex has an enlarged targetbinding surface area as compared to the free drug such that at least oneof enhanced affinity, specificity or selectivity are observed ascompared to the free drug. In a first embodiment in which increasedaffinity is observed, the binary complex binds to the target to form atripartite complex characterized by the presence of presenter-targetbinding interactions as well as drug-target binding interactions. Thesubject methods and compositions find use in a variety of therapeuticapplications. In further describing the subject invention, thebifunctional molecules and methods for their production will bedescribed first, followed by a discussion of applications in which thebifunctional molecules find use.

[0037] Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

[0038] In this specification and the appended claims, the singular forms“a,” “an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

[0039] Though not wishing to be bound by any particular theory, thesubject invention provides a means for improving at least one of theaffinity or specificity or selectivity of a small molecule drug for itsdesired target by enlarging the target binding surface area of the drugmoiety as compared to the free drug. Enhanced affinity, specificity orselectivity of the drug is accomplished by presenting it to its drugtarget as a binary complex made up of a bifunctional molecule of thedrug and presenter protein ligand bound to a presenter protein.Interactions between the presenter protein and the drug target, such asfavorable interactions, neutral interactions or repulsive interactions,in combination with interactions between the drug moiety and its target,result in a modulation of the overall binding profile of the drug moietyfor its various targets, as compared to a free drug control. As such, byadministering a small molecule drug as a bifunctional molecule accordingto the subject invention, one can achieve improved results as comparedto the results obtainable by administration of the small molecule drugby itself. See FIG. 1.

[0040] Bifunctional Molecule

[0041] A critical element of the subject invention is the bifunctionalmolecule. The bifunctional molecule is a non-naturally occurring orsynthetic compound. The bifunctional molecule is further characterizedin that the presenter protein ligand and the drug moiety are different,such that the bifuntional molecule may be viewed as a heterodimericcompound produced by the joining of two different moieties. In manyembodiments, the presenter protein ligand and the drug moiety are chosensuch that the corresponding drug target and presenter protein do notnaturally associate with each other to produce a biological effect. Inmany preferred embodiments, the bifunctional molecules are capable ofsimultaneously binding two distinct compounds, i.e. a target and apresenter protein, to form a tripartite complex. The bifunctionalmolecule has a drug moiety bonded to a ligand for a presenter protein,either directly or through a linking group. The molecular weight of thebifunctional molecule is generally at least about 100 D, usually atleast about 400 D and more usually at least about 500 D, and may be asgreat as 2000 D or greater, but usually does not exceed about 5000 D.

[0042] The bifunctional molecule is further characterized in that thedrug moiety has improved activity as compared to free drug. By improvedactivity is meant that the drug moiety has a more desirable effect withrespect to the condition being treated, as compared to the correspondingfree drug from which the drug moiety of the bifunctional molecule isderived. In many embodiments, the bifunctional molecule is characterizedby having improved affinity for its target as compared to itscorresponding drug, i.e. a control. The magnitude of enhanced affinityand/or specificity will be at least about 2 fold, usually at least about5 fold and in many embodiments at least 10 fold. In many embodiments,the affinity of the bifunctional molecule for its target will be atleast about 10⁻⁴ M, usually at least about 10⁻⁶ M. Additionally and/oralternatively, the bifunctional molecule exhibits improved specificityfor its target as compared to a free drug control. Additionally and/oralternatively, the bifunctional molecule exhibits improved selectivelyfor its target as compared to a free drug control.

[0043] Bifunctional molecules are generally described by the formula:

Z—L—X

[0044] wherein X is a drug moiety;

[0045] L is bond or linking group; and

[0046] Z is a ligand for an endogenous presenter protein; with theproviso that X and Z are different.

[0047] Drug Moiety: X

[0048] The drug moiety X may be any molecule, as well as binding portionor fragment thereof, that is capable of modulating a biological processin a living host, either by itself or in the context of the presenterprotein/bifunctional molecule binary complex. Generally, X is a smallorganic molecule that is capable of binding to the target of interest.As the drug moiety of the bifunctional molecule is a small molecule, itgenerally has a molecular weight of at least about 50 D, usually atleast about 100 D, where the molecular weight may be as high as 500 D orhigher, but will usually not exceed about 2000 D.

[0049] The drug moiety is capable of interacting with a target in thehost into which the bifunctional molecule is administered duringpractice of the subject methods. The target may be a number of differenttypes of naturally occurring structures, where targets of interestinclude both intracellular and extracellular targets, where such targetsmay be proteins, phospholipids, nucleic acids and the like, whereproteins are of particular interest. Specific proteinaceous targets ofinterest include, without limitation, enzymes, e.g. kinases,phosphatases, reductases, cyclooxygenases, proteases and the like,targets comprising domains involved in protein-protein interactions,such as the SH2, SH3, PTB and PDZ domains, structural proteins, e.g.actin, tubulin, etc., membrane receptors, immunoglobulins, e.g. IgE,cell adhesion receptors, such as integrins, etc, ion channels,transmembrane pumps, transcription factors, signaling proteins, and thelike.

[0050] The drug moiety of the bifunctional compound will include one ormore functional groups necessary for structural interaction with thetarget, e.g. groups necessary for hydrophobic, hydrophilic,electrostatic or even covalent interactions, depending on the particulardrug and its intended target. Where the target is a protein, the drugmoiety will include functional groups necessary for structuralinteraction with proteins, such as hydrogen bonding,hydrophobic-hydrophobic interactions, electrostatic interactions, etc.,and will typically include at least an amine, amide, sulfhydryl,carbonyl, hydroxyl or carboxyl group, preferably at least two of thefunctional chemical groups. As described in greater detail below, thedrug moiety will also comprise a region that may be modified and/orparticipate in covalent linkage to the other components of thebifunctional molecule, such as the presenter protein ligand or linker,without substantially adversely affecting the moiety's ability to bindto its target.

[0051] The drug moieties often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Also of interest as drugmoieties are structures found among biomolecules, including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Such compounds may bescreened to identify those of interest, where a variety of differentscreening protocols are known in the art.

[0052] The drug moiety of the bifunctional molecule may be derived froma naturally occurring or synthetic compound that may be obtained from awide variety of sources, including libraries of synthetic or naturalcompounds. For example, numerous means are available for random anddirected synthesis of a wide variety of organic compounds andbiomolecules, including the preparation of randomized oligonucleotidesand oligopeptides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means, and may be used to producecombinatorial libraries. Known pharmacological agents may be subjectedto directed or random chemical modifications, such as acylation,alkylation, esterification, amidification, etc. to produce structuralanalogs.

[0053] As such, the drug moiety may be obtained from a library ofnaturally occurring or synthetic molecules, including a library ofcompounds produced through combinatorial means, i.e. a compounddiversity combinatorial library. When obtained from such libraries, thedrug moiety employed will have demonstrated some desirable activity inan appropriate screening assay for the activity. Combinatoriallibraries, as well as methods for the production and screening, areknown in the art and described in: U.S. Pat. Nos. 5,741,713; 5,734,018;5,731,423; 5,721,099; 5,708,153; 5,698,673; 5,688,997; 5,688,696;5,684,711; 5,641,862; 5,639,603; 5,593,853; 5,574,656; 5,571,698;5,565,324; 5,549,974; 5,545,568; 5,541,061; 5,525,735; 5,463,564;5,440,016; 5,438,119; 5,223,409, the disclosures of which are hereinincorporated by reference.

[0054] Specific drugs of interest from which the drug moiety may bederived include, but are not limited to: psychopharmacological agents,such as (1) central nervous system depressants, e.g. general anesthetics(barbiturates, benzodiazepines, steroids, cyclohexanone derivatives, andmiscellaneous agents), sedative-hypnotics (benzodiazepines,barbiturates, piperidinediones and triones, quinazoline derivatives,carbamates, aldehydes and derivatives, amides, acyclic ureides,benzazepines and related drugs, phenothiazines, etc.), central voluntarymuscle tone modifying drugs (anticonvulsants, such as hydantoins,barbiturates, oxazolidinediones, succinimides, acylureides,glutarimides, benzodiazepines, secondary and tertiary alcohols,dibenzazepine derivatives, valproic acid and derivatives, GABA analogs,etc.), analgesics (morphine and derivatives, oripavine derivatives,morphinan derivatives, phenylpiperidines, 2,6-methane-3-benzazocainederivatives, diphenylpropylamines and isosteres, salicylates,p-aminophenol derivatives, 5-pyrazolone derivatives, arylacetic acidderivatives, fenamates and isosteres, etc.) and antiemetics(anticholinergics, antihistamines, antidopaminergics, etc.), (2) centralnervous system stimulants, e.g. analeptics (respiratory stimulants,convulsant stimulants, psychomotor stimulants), narcotic antagonists(morphine derivatives, oripavine derivatives, 2,6-methane-3-benzoxacinederivatives, morphinan derivatives) nootropics, (3)psychopharmacologicals, e.g. anxiolytic sedatives (benzodiazepines,propanediol carbamates) antipsychotics (phenothiazine derivatives,thioxanthine derivatives, other tricyclic compounds, butyrophenonederivatives and isosteres, diphenylbutylamine derivatives, substitutedbenzamides, arylpiperazine derivatives, indole derivatives, etc.),antidepressants (tricyclic compounds, MAO inhibitors, etc.), (4)respiratory tract drugs, e.g. central antitussives (opium alkaloids andtheir derivatives);

[0055] pharmacodynamic agents, such as (1) peripheral nervous systemdrugs, e.g. local anesthetics (ester derivatives, amide derivatives),(2) drugs acting at synaptic or neuroeffector junctional sites, e.g.cholinergic agents, cholinergic blocking agents, neuromuscular blockingagents, adrenergic agents, antiadrenergic agents, (3) smooth muscleactive drugs, e.g. spasmolytics (anticholinergics, musculotropicspasmolytics), vasodilators, smooth muscle stimulants, (4) histaminesand antihistamines, e.g. histamine and derivative thereof (betazole),antihistamines (H₁-antagonists, H₂-antagonists), histamine metabolismdrugs, (5) cardiovascular drugs, e.g. cardiotonics (plant extracts,butenolides, pentadienolids, alkaloids from erythrophleum species,ionophores, -adrenoceptor stimulants, etc), antiarrhythmic drugs,antihypertensive agents, antilipidemic agents (clofibric acidderivatives, nicotinic acid derivatives, hormones and analogs,antibiotics, salicylic acid and derivatives), antivaricose drugs,hemostyptics, (6) blood and hemopoietic system drugs, e.g. antianemiadrugs, blood coagulation drugs (hemostatics, anticoagulants,antithrombotics, thrombolytics, blood proteins and their fractions), (7)gastrointestinal tract drugs, e.g. digestants (stomachics, choleretics),antiulcer drugs, antidiarrheal agents, (8) locally acting drugs;

[0056] chemotherapeutic agents, such as (1) anti-infective agents, e.g.ectoparasiticides (chlorinated hydrocarbons, pyrethins, sulfuratedcompounds), anthelmintics, antiprotozoal agents, antimalarial agents,antiamebic agents, antileiscmanial drugs, antitrichomonal agents,antitrypanosomal agents, sulfonamides, antimycobacterial drugs,antiviral chemotherapeutics, etc., and (2) cytostatics, i.e.antineoplastic agents or cytotoxic drugs, such as alkylating agents,e.g. Mechlorethamine hydrochloride (Nitrogen Mustard, Mustargen, HN2),Cyclophosphamide (Cytovan, Endoxana), Ifosfamide (IFEX), Chlorambucil(Leukeran), Melphalan (Phenylalanine Mustard, L-sarcolysin, Alkeran,L-PAM), Busulfan (Myleran), Thiotepa (Triethylenethiophosphoramide),Carmustine (BiCNU, BCNU), Lomustine (CeeNU, CCNU), Streptozocin(Zanosar) and the like; plant alkaloids, e.g. Vincristine (Oncovin),Vinblastine (Velban, Velbe), Paclitaxel (Taxol), and the like;antimetabolites, e.g. Methotrexate (MTX), Mercaptopurine (Purinethol,6-MP), Thioguanine (6-TG), Fluorouracil (5-FU), Cytarabine (Cytosar-U,Ara-C), Azacitidine (Mylosar, 5-AZA) and the like; antibiotics, e.g.Dactinomycin (Actinomycin D, Cosmegen), Doxorubicin (Adriamycin),Daunorubicin (duanomycin, Cerubidine), Idarubicin (Idamycin), Bleomycin(Blenoxane), Picamycin (Mithramycin, Mithracin), Mitomycin (Mutamycin)and the like, and other anticellular proliferative agents, e.g.Hydroxyurea (Hydrea), Procarbazine (Mutalane), Dacarbazine (DTIC-Dome),Cisplatin (Platinol) Carboplatin (Paraplatin), Asparaginase (Elspar)Etoposide (VePesid, VP-16-213), Amsarcrine (AMSA, m-AMSA), Mitotane(Lysodren), Mitoxantrone (Novatrone), and the like;

[0057] Antibiotics, such as: aminoglycosides, e.g. amikacin, apramycin,arbekacin, bambermycins, butirosin, dibekacin, dihydrostreptomycin,fortimicin, gentamicin, isepamicin, kanamycin, micronomcin, neomycin,netilmicin, paromycin, ribostamycin, sisomicin, spectinomycin,streptomycin, tobramycin, trospectomycin; amphenicols, e.g.azidamfenicol, chloramphenicol, florfenicol, and theimaphenicol;ansamycins, e.g. rifamide, rifampin, rifamycin, rifapentine, rifaximin;β-lactams, e.g. carbacephems, carbapenems, cephalosporins, cehpamycins,monobactams, oxaphems, penicillins; lincosamides, e.g. clinamycin,lincomycin; macrolides, e.g. clarithromycin, dirthromycin, erythromycin,etc.; polypeptides, e.g. amphomycin, bacitracin, capreomycin, etc.;tetracyclines, e.g. apicycline, chlortetracycline, clomocycline, etc.;synthetic antibacterial agents, such as 2,4-diaminopyrimidines,nitrofurans, quinolones and analogs thereof, sulfonamides, sulfones;

[0058] Antifungal agents, such as: polyenes, e.g. amphotericin B,candicidin, dennostatin, filipin, fungichromin, hachimycin, hamycin,lucensomycin, mepartricin, natamycin, nystatin, pecilocin, perimycin;synthetic antifungals, such as allylamines, e.g. butenafine, naftifine,terbinafine; imidazoles, e.g. bifonazole, butoconazole, chlordantoin,chlormidazole, etc., thiocarbamates, e.g. tolciclate, triazoles, e.g.fluconazole, itraconazole, terconazole;

[0059] Anthelmintics, such as: arecoline, aspidin, aspidinol,dichlorophene, embelin, kosin, napthalene, niclosamide, pelletierine,quinacrine, alantolactone, amocarzine, amoscanate, ascaridole,bephenium, bitoscanate, carbon tetrachloride, carvacrol, cyclobendazole,diethylcarbamazine, etc.;

[0060] Antimalarials, such as: acedapsone, amodiaquin, arteether,artemether, artemisinin, artesunate, atovaquone, bebeerine, berberine,chirata, chlorguanide, chloroquine, chlorprogaunil, cinchona,cinchonidine, cinchonine, cycloguanil, gentiopicrin, halofantrine,hydroxychloroquine, mefloquine hydrochloride, 3-methylarsacetin,pamaquine, plasmocid, primaquine, pyrimethamine, quinacrine, quinidine,quinine, quinocide, quinoline, dibasic sodium arsenate;

[0061] Antiprotozoan agents, such as: acranil, tinidazole, ipronidazole,ethylstibamine, pentamidine, acetarsone, aminitrozole, anisomycin,nifuratel, tinidazole, benzidazole, suramin, and the like.

[0062] Name brand drugs of interest include, but are not limited to:Rezulin™, Lovastatin™, Enalapril™, Prozac™, Prilosec™, Lipotor™,Claritin™, Zocor™, Ciprofloxacin™, Viagra™, Crixivan™, Ritalin™, and thelike.

[0063] Drug compounds of interest from which drug moieties may bederived are also listed in: Goodman & Gilman's, The PharmacologicalBasis of Therapeutics (9th Ed) (Goodman et al. eds) (McGraw-Hill)(1996); and 1999 Physician's Desk Reference (1998).

[0064] Specific compounds of interest also include, but are not limitedto:

[0065] antineoplastic agents, as disclosed in U.S. Pat. Nos. 5,880,161,5,877,206, 5,786,344, 5,760,041, 5,753,668, 5,698,529, 5,684,004,5,665,715, 5,654,484, 5,624,924, 5,618,813, 5,610,292, 5,597,831,5,530,026, 5,525,633, 5,525,606, 5,512,678, 5,508,277, 5,463,181,5,409,893, 5,358,952, 5,318,965, 5,223,503, 5,214,068, 5,196,424,5,109,024, 5,106,996, 5,101,072, 5,077,404, 5,071,848, 5,066,493,5,019,390, 4,996,229, 4,996,206, 4,970,318, 4,968,800, 4,962,114,4,927,828, 4,892,887, 4,889,859, 4,886,790, 4,882,334, 4,882,333,4,871,746, 4,863,955, 4,849,563, 4,845,216, 4,833,145, 4,824,955,4,785,085, 4,76,925, 4,684,747, 4,618,685, 4,611,066, 4,550,187,4,550,186, 4,544,501, 4,541,956, 4,532,327, 4,490,540, 4,399,283,4,391,982, 4,383,994, 4,294,763, 4,283,394, 4,246,411, 4,214,089,4,150,231, 4,147,798, 4,056,673, 4,029,661, 4,012,448;

[0066] psycopharmacological/psychotropic agents, as disclosed in U.S.Pat. Nos. 5,192,799, 5,036,070, 4,778,800, 4,753,951, 4,590,180,4,690,930, 4,645,773, 4,427,694, 4,424,202, 4,440,781, 5,686,482,5,478,828, 5,461,062, 5,387,593, 5,387,586, 5,256,664, 5,192,799,5,120,733, 5,036,070, 4,977,167, 4,904,663, 4,788,188, 4,778,800,4,753,951, 4,690,930, 4,645,773, 4,631,285, 4,617,314, 4,613,600,4,590,180, 4,560,684, 4,548,938, 4,529,727, 4,459,306, 4,443,451,4,440,781, 4,427,694, 4,424,202, 4,397,853, 4,358,451, 4,324,787,4,314,081, 4,313,896, 4,294,828, 4,277,476, 4,267,328, 4,264,499,4,231,930, 4,194,009, 4,188,388, 4,148,796, 4,128,717, 4,062,858,4,031,226, 4,020,072, 4,018,895, 4,018,779, 4,013,672, 3,994,898,3,968,125, 3,939,152, 3,928,356, 3,880,834, 3,668,210;

[0067] cardiovascular agents, as disclosed in U.S. Pat. No. 4,966,967,5,661,129, 5,552,411, 5,332,737, 5,389,675, 5,198,449, 5,079,247,4,966,967, 4,874,760, 4,954,526, 5,051,423, 4,888,335, 4,853,391,4,906,634, 4,775,757, 4,727,072, 4,542,160, 4,522,949, 4,524,151,4,525,479, 4,474,804, 4,520,026, 4,520,026, 5,869,478, 5,859,239,5,837,702, 5,807,889, 5,731,322, 5,726,171, 5,723,457, 5,705,523,5,696,111, 5,691,332, 5,679,672, 5,661,129, 5,654,294, 5,646,276,5,637,586, 5,631,251, 5,612,370, 5,612,323, 5,574,037, 5,563,170,5,552,411, 5,552,397, 5,547,966, 5,482,925, 5,457,118, 5,414,017,5,414,013, 5,401,758, 5,393,771, 5,362,902, 5,332,737, 5,310,731,5,260,444, 5,223,516, 5,217,958, 5,208,245, 5,202,330, 5,198,449,5,189,036, 5,185,362, 5,140,031, 5,128,349, 5,116,861, 5,079,247,5,070,099, 5,061,813, 5,055,466, 5,051,423, 5,036,065, 5,026,712,5,011,931, 5,006,542, 4,981,843, 4,977,144, 4,971,984, 4,966,967,4,959,383, 4,954,526, 4,952,692, 4,939,137, 4,906,634, 4,889,866,4,888,335, 4,883,872, 4,883,811, 4,847,379, 4,835,157, 4,824,831,4,780,538, 4,775,757, 4,774,239, 4,771,047, 4,769,371, 4,767,756,4,762,837, 4,753,946, 4,752,616, 4,749,715, 4,738,978, 4,735,962,4,734,426, 4,734,425, 4,734,424, 4,730,052, 4,727,072, 4,721,796,4,707,550, 4,704,382, 4,703,120, 4,681,970, 4,681,882, 4,670,560,4,670,453, 4,668,787, 4,663,337, 4,663,336, 4,661,506, 4,656,267,4,656,185, 4,654,357, 4,654,356, 4,654,355, 4,654,335, 4,652,578,4,652,576, 4,650,874, 4,650,797, 4,649,139, 4,647,585, 4,647,573,4,647,565, 4,647,561, 4,645,836, 4,639,461, 4,638,012, 4,638,011,4,632,931, 4,631,283, 4,628,095, 4,626,548, 4,614,825, 4,611,007,4,611,006, 4,611,005, 4,609,671, 4,608,386, 4,607,049, 4,607,048,4,595,692, 4,593,042, 4,593,029, 4,591,603, 4,588,743, 4,588,742,4,588,741, 4,582,854, 4,575,512, 4,568,762, 4,560,698, 4,556,739,4,556,675, 4,555,571, 4,555,570, 4,555,523, 4,550,120, 4,542,160,4,542,157, 4,542,156, 4,542,155, 4,542,151, 4,537,981, 4,537,904,4,536,514, 4,536,513, 4,533,673, 4,526,901, 4,526,900, 4,525,479,4,524,151, 4,522,949, 4,521,539, 4,520,026, 4,517,188, 4,482,562,4,474,804, 4,474,803, 4,472,411, 4,466,979, 4,463,015, 4,456,617,4,456,616, 4,456,615, 4,418,076, 4,416,896, 4,252,815, 4,220,594,4,190,587, 4,177,280, 4,164,586, 4,151,297, 4,145,443, 4,143,054,4,123,550, 4,083,968, 4,076,834, 4,064,259, 4,064,258, 4,064,257,4,058,620, 4,001,421, 3,993,639, 3,991,057, 3,982,010, 3,980,652,3,968,117, 3,959,296, 3,951,950, 3,933,834, 3,925,369, 3,923,818,3,898,210, 3,897,442, 3,897,441, 3,886,157, 3,883,540, 3,873,715,3,867,383, 3,873,715, 3,867,383, 3,691,216, 3,624,126;

[0068] antimicrobial agents as disclosed in U.S. Pat. Nos. 5,902,594,5,874,476, 5,874,436, 5,859,027, 5,856,320, 5,854,242, 5,811,091,5,786,350, 5,783,177, 5,773,469, 5,762,919, 5,753,715, 5,741,526,5,709,870, 5,707,990, 5,696,117, 5,684,042, 5,683,709, 5,656,591,5,643,971, 5,643,950, 5,610,196, 5,608,056, 5,604,262, 5,595,742,5,576,341, 5,554,373, 5,541,233, 5,534,546, 5,534,508, 5,514,715,5,508,417, 5,464,832, 5,428,073, 5,428,016, 5,424,396, 5,399,553,5,391,544, 5,385,902, 5,359,066, 5,356,803, 5,354,862, 5,346,913,5,302,592, 5,288,693, 5,266,567, 5,254,685, 5,252,745, 5,209,930,5,196,441, 5,190,961, 5,175,160, 5,157,051, 5,096,700, 5,093,342,5,089,251, 5,073,570, 5,061,702, 5,037,809, 5,036,077, 5,010,109,4,970,226, 4,916,156, 4,888,434, 4,870,093, 4,855,318, 4,784,991,4,746,504, 4,686,221, 4,599,228, 4,552,882, 4,492,700, 4,489,098,4,489,085, 4,487,776, 4,479,953, 4,477,448, 4,474,807, 4,470,994,4,370,484, 4,337,199, 4,311,709, 4,308,283, 4,304,910, 4,260,634,4,233,311, 4,215,131, 4,166,122, 4,141,981, 4,130,664, 4,089,977,4,089,900, 4,069,341, 4,055,655, 4,049,665, 4,044,139, 4,002,775,3,991,201, 3,966,968, 3,954,868, 3,936,393, 3,917,476, 3,915,889,3,867,548, 3,865,748, 3,867,548, 3,865,748, 3,783,160, 3,764,676,3,764,677;

[0069] anti-inflammatory agents as disclosed in U.S. Pat. Nos.5,872,109, 5,837,735, 5,827,837, 5,821,250, 5,814,648, 5,780,026,5,776,946, 5,760,002, 5,750,543, 5,741,798, 5,739,279, 5,733,939,5,723,481, 5,716,967, 5,688,949, 5,686,488, 5,686,471, 5,686,434,5,684,204, 5,684,041, 5,684,031, 5,684,002, 5,677,318, 5,674,891,5,672,620, 5,665,752, 5,656,661, 5,635,516, 5,631,283, 5,622,948,5,618,835, 5,607,959, 5,593,980, 5,593,960, 5,580,888, 5,552,424,5,552,422, 5,516,764, 5,510,361, 5,508,026, 5,500,417, 5,498,405,5,494,927: 5,476,876, 5,472,973, 5,470,885, 5,470,842, 5,464,856,5,464,849, 5,462,952, 5,459,151, 5,451,686, 5,444,043, 5,436,265,5,432,181, RE034,918, 5,393,756, 5,380,738, 5,376,670, 5,360,811,5,354,768, 5,348,957, 5,347,029, 5,340,815, 5,338,753, 5,324,648,5,319,099, 5,318,971, 5,312,821, 5,302,597, 5,298,633, 5,298,522,5,298,498, 5,290,800, 5,290,788, 5,284,949, 5,280,045, 5,270,319,5,266,562, 5,256,680, 5,250,700, 5,250,552, 5,248,682, 5,244,917,5,240,929, 5,234,939, 5,234,937, 5,232,939, 5,225,571, 5,225,418,5,220,025, 5,212,189, 5,212,172, 5,208,250, 5,204,365, 5,202,350,5,196,431, 5,191,084, 5,187,175, 5,185,326, 5,183,906, 5,177,079,5,171,864, 5,169,963, 5,155,122, 5,143,929, 5,143,928, 5,143,927,5,124,455, 5,124,347, 5,114,958, 5,112,846, 5,104,656, 5,098,613,5,095,037, 5,095,019, 5,086,064, 5,081,261, 5,081,147, 5,081,126,5,075,330, 5,066,668, 5,059,602, 5,043,457, 5,037,835, 5,037,811,5,036,088, 5,013,850, 5,013,751, 5,013,736, 500,654, 4,992,448,4,992,447, 4,988,733, 4,988,728, 4,981,865, 4,962,119, 4,959,378,4,954,519, 4,945,099, 4,942,236, 4,931,457, 4,927,835, 4,912,248,4,910,192, 4,904,786, 4,904,685, 4,904,674, 4,904,671, 4,897,397,4,895,953, 4,891,370, 4,870,210, 4,859,686, 4,857,644, 4,853,392,4,851,412, 4,847,303, 4,847,290, 4,845,242, 4,835,166, 4,826,990,4,803,216, 4,801,598, 4,791,129, 4,788,205, 4,778,818, 4,775,679,4,772,703, 4,767,776, 4,764,525, 4,760,051, 4,748,153, 4,725,616,4,721,712, 4,713,393, 4,708,966, 4,695,571, 4,686,235, 4,686,224,4,680,298, 4,678,802, 4,652,564, 4,644,005, 4,632,923, 4,629,793,4,614,741, 4,599,360, 4,596,828, 4,595,694, 4,595,686, 4,594,357,4,585,755, 4,579,866, 4,578,390, 4,569,942, 4,567,201, 4,563,476,4,559,348, 4,558,067, 4,556,672, 4,556,669, 4,539,326, 4,537,903,4,536,503, 4,518,608, 4,514,415, 4,512,990, 4,501,755, 4,495,197,4,493,839, 4,465,687, 4,440,779, 4,440,763, 4,435,420, 4,412,995,4,400,534, 4,355,034, 4,335,141, 4,322,420, 4,275,064, 4,244,963,4,235,908, 4,234,593, 4,226,887, 4,201,778, 4,181,720, 4,173,650,4,173,634, 4,145,444, 4,128,664, 4,125,612, 4,124,726, 4,124,707,4,117,135, 4,027,031, 4,024,284, 4,021,553, 4,021,550, 4,018,923,4,012,527, 4,011,326, 3,998,970, 3,998,954, 3,993,763, 3,991,212,3,984,405, 3,978,227, 3,978,219, 3,978,202, 3,975,543, 3,968,224,3,959,368, 3,949,082, 3,949,081, 3,947,475, 3,936,450, 3,934,018,3,930,005, 3,857,955, 3,856,962, 3,821,377, 3,821,401, 3,789,121,3,789,123, 3,726,978, 3,694,471, 3,691,214, 3,678,169, 3,624,216;

[0070] immunosuppressive agents, as disclosed in U.S. Pat. Nos.4,450,159, 4,450,159, 5,905,085, 5,883,119, 5,880,280, 5,877,184,5,874,594, 5,843,452, 5,817,672, 5,817,661, 5,817,660, 5,801,193,5,776,974, 5,763,478, 5,739,169, 5,723,466, 5,719,176, 5,696,156,5,695,753, 5,693,648, 5,693,645, 5,691,346, 5,686,469, 5,686,424,5,679,705, 5,679,640, 5,670,504, 5,665,774, 5,665,772, 5,648,376,5,639,455, 5,633,277, 5,624,930, 5,622,970, 5,605,903, 5,604,229,5,574,041, 5,565,560, 5,550,233, 5,545,734, 5,540,931, 5,532,248,5,527,820, 5,516,797, 5,514,688, 5,512,687, 5,506,233, 5,506,228,5,494,895, 5,484,788, 5,470,857, 5,464,615, 5,432,183, 5,431,896,5,385,918, 5,349,061, 5,344,925, 5,330,993, 5,308,837, 5,290,783,5,290,772, 5,284,877, 5,284,840, 5,273,979, 5,262,533, 5,260,300,5,252,732, 5,250,678, 5,247,076, 5,244,896, 5,238,689, 5,219,884,5,208,241, 5,208,228, 5,202,332, 5,192,773, 5,189,042, 5,169,851,5,162,334, 5,151,413, 5,149,701, 5,147,877, 5,143,918, 5,138,051,5,093,338, 5,091,389, 5,068,323, 5,068,247, 5,064,835, 5,061,728,5,055,290, 4,981,792, 4,810,692, 4,410,696, 4,346,096, 4,342,769,4,317,825, 4,256,766, 4,180,588, 4,000,275, 3,759,921;

[0071] analgesic agents, as disclosed in U.S. Pat. Nos. 5,292,736,5,688,825, 5,554,789, 5,455,230, 5,292,736, 5,298,522, 5,216,165,5,438,064, 5,204,365, 5,017,578, 4,906,655, 4,906,655, 4,994,450,4,749,792, 4,980,365, 4,794,110, 4,670,541, 4,737,493, 4,622,326,4,536,512, 4,719,231, 4,533,671, 4,552,866, 4,539,312, 4,569,942,4,681,879, 4,511,724, 4,556,672, 4,721,712, 4,474,806, 4,595,686,4,440,779, 4,434,175, 4,608,374, 4,395,402, 4,400,534, 4,374,139,4,361,583, 4,252,816, 4,251,530, 5,874,459, 5,688,825, 5,554,789,5,455,230, 5,438,064, 5,298,522, 5,216,165, 5,204,365, 5,030,639,5,017,578, 5,008,264, 4,994,450, 4,980,365, 4,906,655, 4,847,290,4,844,907, 4,794,110, 4,791,129, 4,774,256, 4,749,792, 4,737,493,4,721,712, 4,719,231, 4,681,879, 4,670,541, 4,667,039, 4,658,037,4,634,708, 4,623,648, 4,622,326, 4,608,374, 4,595,686, 4,594,188,4,569,942, 4,556,672, 4,552,866, 4,539,312, 4,536,512, 4,533,671,4,511,724, 4,440,779, 4,434,175, 4,400,534, 4,395,402, 4,391,827,4,374,139, 4,361,583, 4,322,420, 4,306,097, 4,252,816, 4,251,530,4,244,955, 4,232,018, 4,209,520, 4,164,514, 4,147,872, 4,133,819,4,124,713, 4,117,012, 4,064,272, 4,022,836, 3,966,944;

[0072] cholinergic agents, as disclosed in U.S. Pat. Nos. 5,219,872,5,219,873, 5,073,560, 5,073,560, 5,346,911, 5,424,301, 5,073,560,5,219,872, 4,900,748, 4,786,648, 4,798,841, 4,782,071, 4,710,508,5,482,938, 5,464,842, 5,378,723, 5,346,911, 5,318,978, 5,219,873,5,219,872, 5,084,281, 5,073,560, 5,002,955, 4,988,710, 4,900,748,4,798,841, 4,786,648, 4,782,071, 4,745,123, 4,710,508;

[0073] adrenergic agents, as disclosed in U.S. Pat. Nos. 5,091,528,5,091,528, 4,835,157, 5,708,015, 5,594,027, 5,580,892, 5,576,332,5,510,376, 5,482,961, 5,334,601, 5,202,347, 5,135,926, 5,116,867,5,091,528, 5,017,618, 4,835,157, 4,829,086, 4,579,867, 4,568,679,4,469,690, 4,395,559, 4,381,309, 4,363,808, 4,343,800, 4,329,289,4,314,943, 4,311,708, 4,304,721, 4,296,117, 4,285,873, 4,281,189,4,278,608, 4,247,710, 4,145,550, 4,145,425, 4,139,535, 4,082,843,4,011,321, 4,001,421, 3,982,010, 3,940,407, 3,852,468, 3,832,470;

[0074] antihistamine agents, as disclosed in U.S. Pat. Nos. 5,874,479,5,863,938, 5,856,364, 5,770,612, 5,702,688, 5,674,912, 5,663,208,5,658,957, 5,652,274, 5,648,380, 5,646,190, 5,641,814, 5,633,285,5,614,561, 5,602,183, 4,923,892, 4,782,058, 4,393,210, 4,180,583,3,965,257, 3,946,022, 3,931,197;

[0075] steroidal agents, as disclosed in U.S. Pat. Nos. 5,863,538,5,855,907, 5,855,866, 5,780,592, 5,776,427, 5,651,987, 5,346,887,5,256,408, 5,252,319, 5,209,926, 4,996,335, 4,927,807, 4,910,192,4,710,495, 4,049,805, 4,004,005, 3,670,079, 3,608,076, 5,892,028,5,888,995, 5,883,087, 5,880,115, 5,869,475, 5,866,558, 5,861,390,5,861,388, 5,854,235, 5,837,698, 5,834,452, 5,830,886, 5,792,758,5,792,757, 5,763,361, 5,744,462, 5,741,787, 5,741,786, 5,733,899,5,731,345, 5,723,638, 5,721,226, 5,712,264, 5,712,263, 5,710,144,5,707,984, 5,705,494, 5,700,793, 5,698,720, 5,698,545, 5,696,106,5,677,293, 5,674,861, 5,661,141, 5,656,621, 5,646,136, 5,637,691,5,616,574, 5,614,514, 5,604,215, 5,604,213, 5,599,807, 5,585,482,5,565,588, 5,563,259, 5,563,131, 5,561,124, 5,556,845, 5,547,949,5,536,714, 5,527,806, 5,506,354, 5,506,221, 5,494,907, 5,491,136,5,478,956, 5,426,179, 5,422,262, 5,391,776, 5,382,661, 5,380,841,5,380,840, 5,380,839, 5,373,095, 5,371,078, 5,352,809, 5,344,827,5,344,826, 5,338,837, 5,336,686, 5,292,906, 5,292,878, 5,281,587,5,272,140, 5,244,886, 5,236,912, 5,232,915, 5,219,879, 5,218,109,5,215,972, 5,212,166, 5,206,415, 5,194,602, 5,166,201, 5,166,055,5,126,488, 5,116,829, 5,108,996, 5,099,037, 5,096,892, 5,093,502,5,086,047, 5,084,450, 5,082,835, 5,081,114, 5,053,404, 5,041,433,5,041,432, 5,034,548, 5,032,586, 5,026,882, 4,996,335, 4,975,537,4,970,205, 4,954,446, 4,950,428, 4,946,834, 4,937,237, 4,921,846,4,920,099, 4,910,226, 4,900,725, 4,892,867, 4,888,336, 4,885,280,4,882,322, 4,882,319, 4,882,315, 4,874,855, 4,868,167, 4,865,767,4,861,875, 4,861,765, 4,861,763, 4,847,014, 4,774,236, 4,753,932,4,711,856, 4,710,495, 4,701,450, 4,701,449, 4,689,410, 4,680,290,4,670,551, 4,664,850, 4,659,516, 4,647,410, 4,634,695, 4,634,693,4,588,530, 4,567,000, 4,560,557, 4,558,041, 4,552,871, 4,552,868,4,541,956, 4,519,946, 4,515,787, 4,512,986, 4,502,989, 4,495,102;

[0076] the disclosures of which are herein incorporated by reference.

[0077] The drug moiety of the bifunctional molecule may be the wholecompound or a binding fragment or portion thereof that retains itsaffinity and specificity for the target of interest while having alinkage site for covalent bonding to the presenter protein ligand orlinker.

[0078] Presenter Protein Ligand: Z

[0079] Z is a ligand for a presenter protein present in the host intowhich the bifunctional molecule is to be administered. The presenterprotein ligand of the subject bifunctional molecules binds to a specificpresenter protein present in the host. The binding interaction betweenthe presenter protein and the presenter protein ligand is non-covalent,such that no covalent bonds are produced between the bifunctionalmolecule and the presenter protein upon binding of the two entities. Thepresenter protein ligand is small, where the size of the presenterprotein ligand does not exceed about 4950 daltons, usually does notexceed about 4925 daltons and more usually does not exceed about 4900daltons, where the size of the presenter protein ligand is generally atleast about 50 daltons and more usually at least about 100 daltons. Thepresenter protein ligand, in the context of the bifunctional molecule,has substantially no pharmacological activity at its effectiveconcentration beyond binding to the presenter protein, i.e. it does notdirectly cause a presenter protein-mediated pharmacological event tooccur upon binding at its effective concentration to the presenterprotein, where a presenter protein-mediated pharmacological event is apharmacologically relevant event which is directly modulated by thepresenter protein in the absence of the subject bifunctional molecules.As used herein, pharmacological event is an event that is distinct froma biochemical event (e.g. inhibition a prolyl isomerase activity) or abiological event (e.g. inducement of a cell to express new genes).

[0080] The presenter protein to which the ligand of the bifunctionalmolecule binds may be any protein that is present in the host at thetime the bifunctional molecule is introduced to the host, i.e. thepresenter protein will be endogenous to the host. The presenter proteinmay or may not have one or more modified residues, e.g. residues thatare glycosylated, such that the presenter protein may or may not be aglycoprotein. Furthermore, the presenter protein that is recruited bythe bifunctional molecule may or may not be part of a complex orstructure of a plurality of biological molecules, e.g. lipids, wheresuch complexes or structures may include lipoproteins, lipid bilayers,and the like. However, in many embodiments, the presenter protein thatis recruited by the presenter protein ligand of the bifunctionalmolecule will be by itself, i.e. will not be part of a larger structureof a plurality of biological molecules. Though the presenter protein maybe a protein that is not native to the host but has been introduced atsome time prior to introduction of the bifunctional molecule, e.g.through prior administration of the protein or a nucleic acidcomposition encoding the same, such as through gene therapy, thepresenter protein will, in many embodiments, be a protein that is nativeto and naturally expressed by at least some of the host's cells, i.e. anaturally occurring protein in the host. The presenter protein is aprotein that is present in the region of host occupied by the drugtarget. As such, where the drug target is an intracellular drug target,the presenter protein will be an intracellular protein present in thecell comprising the target, typically expressed in the cell comprisingthe target, i.e. the presenter protein and target are co-expressed inthe same cell. Likewise, where the drug target is an extracellular drugtarget, the presenter protein will be an extracellular protein that isfound in the vicinity of the target.

[0081] Although not a requirement in certain embodiments, in manypreferred embodiments the presenter protein is one that is present inthe host in sufficient quantities such that, upon binding of at least aportion of presenter protein present in the host to the bifunctionalmolecule, adverse pharmacological effects do not occur. In other words,the presenter protein in these preferred embodiments is one in which itsnative and desirable biological activity, if any, is not diminished byan unacceptable amount following binding of the portion of the presenterprotein population to the bifunctional molecule. The amount ofdiminished activity of the presenter protein that is acceptable in agiven situation is determined with respect to the condition beingtreated in view of the benefits of treatment versus the reduction ofoverall presenter protein activity, if any. In certain situations, alarge decrease in overall presenter protein activity may be acceptable,e.g. where the presenter protein activity aggravates the condition beingtreated.

[0082] Specific presenter proteins of interest include intracellular andextracellular proteins. Intracellular proteins of interest include:peptidyl-prolyl isomerases, e.g. FKBPs and cyclophilins; ubiquitouslyexpressed molecular chaperones, e.g. Heat Shock Protein 90 (Hsp90);steroid hormone receptors, e.g. estrogen receptors, glucocorticoidreceptors, androgen receptors; retinoic acid binding protein,cytoskeletal proteins, such as tubulin and actin; etc.

[0083] Of particular interest as intracellular presenter proteins arecis-trans peptidyl-prolyl isomerases which interact with many proteinsbecause of their chaperonin/isomerase activity, e.g. FKBPs andcyclophilins. Peptidyl-prolyl isomerases of interest include FKBPs. Anumber of different FKBPs are known in the art, and include thosedescribed in: Sabatini et al., Mol. Neurobiol. (Oct. 1997) 15:223-239;Marks, Physiol. Rev. (July 1996) 76:631-649; Kay, Biochem J. (March,1996) 314: 361-385; Braun et al., FASEB J. (January 1995) 9:63-72;Fruman et al, FASEB J. (April 1994) 8:391-400; and Hacker et al., Mol.Microbiol. (November 1993) 10: 445-456. FKBPs of interest include FKBP12, FKBP 52, FKBP 14.6 (described in U.S. Pat. No. 5,525,523, thedisclosure of which is herein incorporated by reference); FKBP 12.6(described in U.S. Pat. No. 5,457,182 the disclosure of which is hereinincorporated by reference); FKBP 13 (described in U.S. Pat. No.5,498,597, the disclosure of which is herein incorporated by reference);and HCB (described in U.S. Pat. No. 5,196,352 the disclosure of which isherein incorporated by reference); where FKBP 12 and FKBP 52 are ofparticular interest as intracellular presenter proteins.

[0084] Also of specific interest as presenter proteins are cyclophilins.A number of cyclophilins are known in the art and are described inTrandinh et al., FASEB J. (December 1992) 6: 3410-3420; Harding et al.,Transplantation (August 1988) 46: 29S-35S. Specific cyclophilins ofinterest as intracellular presenter proteins include cyclophilin A, B,C, D, E, and the like, where cyclophilin A is of particular interest.

[0085] Instead of being an intracellular protein, the endogenouspresenter protein may be an extracellular or serum protein. Serumpresenter proteins of particular interest are those that are relativelyabundant in the serum of the host and meet the above criteria forsuitable endogenous presenter proteins. By relatively abundant is meantthat the concentration of the serum presenter protein is at least about1 ng/ml, usually at least about 10 g/ml and more usually at least about15 g/ml. Specific serum proteins of interest as presenter proteinsinclude: albumin, Vitamin A binding proteins and Vitamin D bindingproteins, -2 macroglobulin, with albumin being a particularly preferredpresenter protein.

[0086] The Z moiety of the subject bifunctional molecules will thereforebe chosen in view of the endogenous presenter protein that is to berecruited to produce the at least binary and, in some embodiments,tripartite complex. As such, the Z moiety may be a number of differentligands, depending on the particular endogenous presenter protein towhich it is intended to bind. In many preferred embodiments, the Zmoiety has an affinity for its presenter protein of at least about 10⁻⁴M, usually at least about 10⁻⁶ molar and more usually at least about10⁻⁸ M, where in many embodiments the Z moiety has an affinity for itspresenter protein of between about 10⁻⁹ and 10⁻¹² M. The Z moietyportion of the bifunctional molecule should also be specific for thepresenter protein in the context of its binding activity when present inthe bifunctional molecule, in that it does not significantly bind orsubstantially affect non-presenter proteins when it is present in thebifunctional molecule.

[0087] Representative ligands capable of serving as the Z moiety of thebifunctional molecule include ligands for intracellular proteins, suchas: peptidyl-prolyl isomerase ligands, e.g. FK506, rapamycin,cyclosporin A and the like; Hsp90 ligands, e.g. geldanamycin; steroidhormone receptor ligands, e.g. naturally occurring steroid hormones,such as estrogen, progestin, testosterone, and the like, as well assynthetic derivatives and mimetics thereof, particularly those whichbind with high specificity and affinity but do not activate theirrespective receptors; small molecules that bind to cytoskeletalproteins, e.g. antimitotic agents, such as taxanes, colchicine,colcemid, nocadozole, vinblastine, and vincristine, actin bindingagents, such as cytochalasin, latrunculin, phalloidin, and the like.

[0088] As mentioned above, the preferred intracellular presenterproteins are members of the peptidyl-prolyl isomerase family,particularly the FKBP and cyclophilin subsets of this family. Wherepeptidyl-prolyl isomerase presenter proteins are employed, thebifunctional molecule/peptidyl-prolyl isomerase complex will preferablynot substantially bind to the natural peptidyl-prolyl isomerase/ligandtarget calcineurin so as to result in significant immunosuppression. Avariety of ligands are known that bind to FKBPs and may be used in thesubject invention. The ligands should specifically bind to an FKBP andhave an affinity for the FKBP that is between about 10⁻⁶ and 10⁻¹⁰ M. Ofinterest are both naturally occurring FKBP ligands, including FK506 andrapamycin. Also of interest are synthetic FKBP ligands, including thosedescribed in U.S. Pat. Nos. 5,665,774; 5,622,970; 5,516,797; 5,614,547;and 5,403,833, the disclosures of which are herein incorporated byreference.

[0089] Also of interest are cyclophilin ligands, where such ligandsshould specifically bind to cyclophilin with an affinity that is betweenabout 10⁻⁶ and 10⁻⁹ M. A variety of ligands that bind to cyclophilinsare also known, where such ligands include the naturally occurringcyclosporins, such as cyclosporin A, as well as synthetic derivativesand mimetics thereof, including those described in U.S. Pat. Nos.5,401,649; 5,318,901; 5,236,899; 5,227,467; 5,214,130; 5,122,511;5,116,816; 5,089,390; 5,079,341; 5,017,597; 4,940,719; 4,914,188;4,885,276; 4,798,823; 4,771,122; 4,703,033; 4,554,351; 4,396,542;4,289,851; 4,288,431; 4,220,61 and 4,210,581, the disclosures of whichare herein incorporated by reference.

[0090] Representative ligands for use as the Z moiety in thebifunctional molecule also include ligands that bind to extracellularpresenter proteins. Such ligands should specifically bind to theirrespective presenter protein with an affinity of at least about 10⁻⁴ M.Ligands of interest for use in binding to extracellular presenterproteins include: albumin ligands, such as arachidonate, bilirubin,hemin, aspirin, ibuprofen, para-amino salicylic acid, myristylate,plamitate, linoleate, warfarin etc.; Vitamin A and derivatives thereof,Vitamin D and derivatives thereof, and the like.

[0091] Linking Moiety: L

[0092] The Z and X moieties of the bifunctional molecule are joinedtogether through linking moiety L, where L may be either a bond or alinking group. Where linking groups are employed, such groups are chosento provide for covalent attachment of the drug and ligand moietiesthrough the linking group, as well as the desired structuralrelationship of the bifunctional molecule with respect to its intendedpresenter protein. Linking groups of interest may vary widely dependingon the nature of the drug and ligand moieties. The linking group, whenpresent, should preferably be biologically inert. Appropriate linkerscan readily be identified using the affinity, specificity or selectivityassays described supra. A variety of linking groups are known to thoseof skill in the art and find use in the subject bifunctional molecules.The linker groups should be sufficiently small so as to provide abifunctional molecule having the overall size characteristics asdescribed above, the size of the linker group, when present, isgenerally at least about 50 daltons, usually at least about 100 daltonsand may be as large as 1000 daltons or larger, but generally will notexceed about 500 daltons and usually will not exceed about 300 daltons.Generally, such linkers will comprise a spacer group terminated ateither end with a reactive functionality capable of covalently bondingto the drug or ligand moieties. Spacer groups of interest possiblyinclude aliphatic and unsaturated hydrocarbon chains, spacers containingheteroatoms such as oxygen (ethers such as polyethylene glycol) ornitrogen (polyamines), peptides, carbohydrates, cyclic or acyclicsystems that may possibly contain heteroatoms. Spacer groups may also becomprised of ligands that bind to metals such that the presence of ametal ion coordinates two or more ligands to form a complex. Specificspacer elements include: 1,4-diaminohexane, xylylenediamine,terephthalic acid, 3,6-dioxaoctanedioic acid,ethylenediamine-N,N-diacetic acid,1,1′-ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid),4,4′-ethylenedipiperidine. Potential reactive functionalities includenucleophilic functional groups (amines, alcohols, thiols, hydrazides),electrophilic functional groups (aldehydes, esters, vinyl ketones,epoxides, isocyanates, maleimides), functional groups capable ofcycloaddition reactions, forming disulfide bonds, or binding to metals.Specific examples include primary and secondary amines, hydroxamicacids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates,oxycarbonylimidazoles, nitrophenylesters, trifluoroethyl esters,glycidyl ethers, vinylsulfones, and maleimides. Specific linker groupsthat may find use in the subject bifunctional molecules includeheterofunctional compounds, such as azidobenzoyl hydrazide,N-[4-(p-azidosalicylamino)butyl]-3′-[2′-pyridyldithio]propionamid),bis-sulfosuccinimidyl suberate, dimethyladipimidate,disuccinimidyltartrate, N-maleimidobutyryloxysuccinimide ester,N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl[4-azidophenyl]-1,3′-dithiopropionate, N-succinimidyl[4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl4-[N-maleimidomethyl]cyclohexane-l-carboxylate,3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP),4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimideester (SMCC), and the like.

[0093] Methods of Making Bifunctional Molecules

[0094] The bifunctional molecules of the subject invention may beprepared using any convenient methodology. In many embodiments of thesubject invention, the invention is used to improve one or more aspectsof an identified and at least partially characterized small moleculedrug. Generally, a small molecule drug of interest but lacking in someof the desired biological activities, such as affinity, specificity orselectivity, is first identified. The drug may be a previouslyidentified biologically active agent or compound having the desiredtarget binding activity, or one that has been newly discovered using oneor more drug discovery techniques. The bifunctional molecule is thengenerally produced from the drug using a rational or combinatorialapproach.

[0095] In a rational approach, the bifunctional molecules areconstructed from their individual components, e.g. ligand, linker anddrug. The components can be covalently bonded to one another throughfunctional groups, as is known in the art, where such functional groupsmay be present on the components or introduced onto the components usingone or more steps, e.g. oxidation reactions, reduction reactions,cleavage reactions and the like. Functional groups that may be used incovalently bonding the components together to produce the bifunctionalmolecule include: hydroxy, sulfhydryl, amino, and the like. Theparticular portion of the different components that are modified toprovide for covalent linkage will be chosen so as not to substantiallyadversely interfere with that components desired binding activity, e.g.for the drug moiety, a region that does not affect the target bindingactivity will be modified, such that a sufficient amount of the desireddrug activity is preserved. Where necessary and/or desired, certainmoieties on the components may be protected using blocking groups, as isknown in the art, see, e.g. Green & Wuts, Protective Groups in OrganicSynthesis (John Wiley & Sons) (1991).

[0096] The above component approach to production of the bifunctionalmolecule is best suited for situations where the crystal structures ofthe presenter protein, ligand, drug and target are known, such thatmolecular modeling can be used to determine the optimal linker size, ifany, to be employed to join the different components.

[0097] Alternatively, the bifunctional molecule can be produced usingcombinatorial methods to produce large libraries of potentialbifunctional molecules which may then be screened for identification ofa bifunctional molecule with the desired binding affinity and/orspecificity. Methods for producing and screening combinatorial librariesof molecules include: U.S. Pat. Nos. 5,741,713; 5,734,018; 5,731,423;5,721,099; 5,708,153; 5,698,673; 5,688,997; 5,688,696; 5,684,711;5,641,862; 5,639,603; 5,593,853; 5,574,656; 5,571,698; 5,565,324;5,549,974; 5,545,568; 5,541,061; 5,525,735; 5,463,564; 5,440,016;5,438,119; 5,223,409, the disclosures of which are herein incorporatedby reference.

[0098] Alternatively, the bifunctional molecule may be produced usingmedicinal chemistry and known structure-activity relationships for thepresenter protein ligand and the drug. In particular, this approach willprovide insight as to where to join the two moieties to the linker.

[0099] Screening Bifunctional Compounds

[0100] The resultant bifunctional molecules are then screened for thosemolecules that exhibit at least one of enhanced affinity, specificity orselectivity as compared to that observed for the free drug. Anyconvenient screening assay may be employed, where the particularscreening assay may be one known to those of skill in the art or onedeveloped in view of the specific molecule and property being studied.Typically, the screening assay will involve observing the bindingactivity of the bifunctional molecule to the target in the presence ofan appropriate presenter protein. For example, where one is interestedin identifying those bifunctional molecules that exhibit enhancedaffinity for their targets as compared to the free drug, one can conductbinding assays and select those bifunctional molecules that exhibitenhanced affinity, where the affinity will generally be at least about 2fold greater than that observed for the free drug, as described above.For specificity, an assay can be used that focuses on the binding of thebifunctional molecule to both desirable and undesirable targets. Forexample, where one is interested in identifying those bifunctionalmolecules that exhibit improved specificity as compared to thecorresponding free drug, where the free drug binds to both desirabletarget A and undesirable target B, one can screen the library for thosebifunctional molecules that, in the presence of presenter protein, bindto target A but with reduced amounts, if at all, to target B, as suchbifunctional molecules are more specific for the desired target thanfree drug. For selectivity, an assay can be used to compare the activityof a bifunctional molecule in the target cell or tissue type to theactivity of the bifunctional molecule in cells or tissues in which drugactivity is not desired. A selective bifunctional molecule will affectthe target in the desired cells, e.g. cells involved in a diseaseprocess, but it will not affect (or at least affect to a lesser extent)the target in undesired cells, e.g. cells not involved in the diseaseprocess. For example, a prospective drug may bind to target A in boththe host and a pathogenic microorganism. By adding a ligand for apresenter protein to the drug, target A in the host is unable to bindthe drug due to interactions with the presenter protein, while the drugretains its potency in the microorganism.

[0101] Methods of Making Bifunctional Molecules for Peptidyl-ProlylIsomerase Presenter Proteins

[0102] As mentioned above, one class of preferred embodiments of thesubject invention are those embodiments in which the bifunctionalmolecules specifically bind to endogenous peptidyl-prolyl isomerasepresenter proteins present in the host into which the bifunctionalmolecule is introduced. Thus, bifunctional molecules of interest includethose in which the endogenous presenter protein is either an FKBP or acyclophilin.

[0103] In preparing bifunctional molecules from FK506, a suitableattachment site on the FK506 structure is identified, modified asnecessary, and then covalently attached to the linker or drug moiety.The structure of FK506 (also known as tacrolimus) is:

[0104] The site to which the linker/drug moiety is covalently attachedis one that, upon covalent attachment, does not ablate the affinityand/or specificity of FK506 for its FKBP presenter protein, e.g. FKBP 12or FKBP 52.As such, positions suitable for use as covalent linkage sitesinclude atoms located between carbon 15 and carbon 25 and thesubstituents attached to these atoms. For example, oxidation of theallyl group or oxidation of the carbon 18 methylene group; modificationof the carbon 22 ketone or the carbon 24 hydroxyl group or alkylation atcarbon 21 or carbon 23; as well as the secondary hydroxyl group locatedon the cyclohexyl ring (carbon 32); are potential specific covalentlinkage sites.

[0105] With FK506, depending on the drug moiety and/or linker to beattached, it may be desirable to introduce one or more functionalmoieties onto the FK506 structure. Functional moieties of interest thatmay be introduced include: hydroxyl groups, amino groups, carboxylgroups, aldehydes, carbonates, carbamates, azides, thiols, and esters,etc. Such groups may be introduced using known protocols, such asoxidation reactions, reduction reactions, cleavage reactions and thelike, with or without the use of one or more blocking groups to preventunwanted side reactions.

[0106] In some instances, it is desirable to covalently attach the drugmoiety directly to FK506, often activated FK506. In such instances, thereactive functional group(s) introduced onto the FK506 structure willdepend primarily on the nature of the drug moiety to be attached. Thus,for peptidic drug moieties, specific pairings of interest include: FK506carbonates for reacting with amino groups of peptides; FK506 carboxylicacids for reacting with amino groups of peptides; FK506 amines forreacting with carboxylic acid groups of peptides; FK506 maleimide forreacting with thiol groups of peptides; and the like. Alternatively,where the drug moiety is a steroid, potential pairings of interestinclude: FK506 N-hydroxysuccinimidyl carbonate and partner amine; FK506aldehyde and partner amine; FK506 aldehyde and partner hydrazide; FK506hydroxy group and partner carboxylic acid OR alkyl halide; FK506 thioland partner maleimide and the like.

[0107] Following introduction of the reactive functional group(s) ontothe FK506 structure, the activated FK506 is then combined with the drugmoiety/linker under conditions sufficient for covalent bonding to occur.

[0108] Another embodiment of particular interest are bifunctionalmolecules of cyclosporin A or analogs thereof. The structure ofcyclosporin A is:

[0109] As with the FK506 bifunctional molecules, the cyclosporin A willbe conjugated to the drug moiety in a manner such that cyclosporin Adoes not substantially lose its affinity for cyclophilin. Preferredpositions on the cyclosporin A structure that may serve as covalentlinkage sites include: residues 4, 5, 6, 7, 8; while less preferred butstill possible residues include: 1, 2, 3, 9, 10 and 11. Where necessary,reactive functionalities may be introduced onto the cyclosporinstructure, where such functionalities include: hydroxyl groups, aminogroups, carboxyl groups, aldehydes, carbonates, carbamates, azides,thiols, and esters, etc., with the particular functionality of interestbeing chosen with respect to the specific linker or drug moiety to beattached.

[0110] Specific Improvements as Compared to Free Drug

[0111] As mentioned above, the bifunctional molecules of the subjectinvention provide for specific improvements over the overall activityobserved in the corresponding free drug, i.e. the bifunctional moleculesexhibit at least one of enhanced affinity, specificity or selectivity ascompared to free drug, i.e. a free drug control. Though not wishing tobe bound to any particular theory, the improved activity as compared tofree drug is thought to result from the enlarged target binding surfacepresent on the binary complex or optimization of favorable interactionsas compared to that found on the free drug. In other words, presentationof the drug as a binary complex of the bifunctional molecule and itspresenter protein modulates the overall binding profile of the drugmoiety with respect to its targets in a way that improves at least oneof the affinity, specificity or selectivity of the drug as compared to afree drug control. This modulation of binding profile can result fromthe combination of drug-target interactions and presenter protein-drugtarget interactions, where the presenter protein-drug targetinteractions may be attractive, repulsive or neutral. See FIG. 1.

[0112]FIG. 1A provides a representation of how enhanced affinity isachieved with the subject invention. As shown, the drug targetestablishes favorable or attractive protein-protein interactions with apresenter protein that presents the drug moiety of the bifunctionalcompound. These favorable interactions between the presenter protein andthe drug target enhance the affinity of the drug for its target. FIG. 1Bprovides a representation of how enhanced specificity is achieved withthe subject bifunctional compounds. In FIG. 1B, a non-specific drugbinding protein (i.e. an undesirable target) cannot bind the drug moietyin the context of the presenter protein because of unfavorableprotein-protein interactions between the presenter protein and thenon-specific drug binding protein. As such, enhanced specificity isobserved since the drug can only bind to its bona fide drug target. FIG.1C provides a representation of how enhanced selectivity is achievedwith the subject bifunctional compounds, where activity of the drug islimited to one type of cell or another within an organism.

[0113] Thus, in a first preferred embodiment, the bifunctional moleculeprovides for enhanced or greater affinity for the target as compared tothe free drug. In preferred embodiments of the subject invention, theenhanced affinity results from positive, i.e. attractive, interactionsbetween the presenter and target as well as between the drug and target.As such, improved affinity results from the production of a tripartitecomplex characterized by the presence of both drug/target bindinginteractions and presenter/target binding interactions.

[0114] In a second preferred embodiment of the subject invention, thebifunctional molecule provides enhanced specificity as compared to thefree drug. In such situations, the bifunctional molecule/presenterprotein binary complex exhibits negative or repulsive interactions withat least some, if not all, of the free drug's targets but not for thedesired target, where any repulsive or negative interactions arisingfrom the presentation of the drug moiety in the context of the presenterprotein are insufficient to prevent a suitable amount of binding of drugto the desired target. Thus, tripartite complexes produced between thebinary complex and undesired targets are, in at least some cases,characterized by the presence of negative or repulsive interactions.Administration of the drug as a bifunctional molecule according to thesubject invention provides for the desired activity, without theundesirable activity that arises from the low specificity of the freedrug.

[0115] In a third embodiment of the subject invention, the bifunctionalmolecule provides for enhanced selectivity as compared to the free drug.In this embodiment, the bifunctional molecule exhibits activity in afirst type of cell but not in a second type of cell. This selectiveactivity is based on the formation of a binary complex between thebifunctional molecule and a presenter protein ligand that is present ina first type of cell but not in a second type of cell. Depending on theparticular bifunctional molecule and drug target, the drug moiety mayonly exhibit activity when presented to the target in the form of abinary complex with the presenter protein. Conversely, the drug moietymay exhibit activity only when presented to the drug target as thebifunctional molecule by itself.

[0116] As such, in certain embodiments, the bifunctional molecule isengineered to have a presenter protein ligand that binds to a presenterprotein present only in those cells that harbor the desired drug target,where activity of the drug moiety is at least enhanced when it ispresented to the drug target by the presenter protein. Those cells whichharbor the undesired target also lack the presenter protein. As such,the drug moiety of the bifunctional molecule exhibits less activity inthese cells since it is not presented by a presenter protein to the drugtarget. In this manner, the activity of the drug moiety has beenselectively enhanced in the first type of cell that comprises thedesired drug target and presenter protein as compared to the second typeof cell that harbors the undesired drug target and lacks the presenterprotein.

[0117] In other embodiments, the drug of interest is one that binds inits free drug state to a desired target in a first type of cell but alsoto an undesired target in a second type of cell. Examples of such drugsinclude antimicrobial agents, such as antibiotics, which bind todesirable targets in microorganisms but undesirable targets in othertypes of cells, such as host cells in which the microorganism ispresent. In this embodiment, the bifunctional molecule is one thatcomprises a ligand for a presenter protein that is present in thosecells comprising the undesirable target(s) but is not present in thosecells harboring the desired target. In other words, the presenter ligandZ of the bifunctional molecule may be any ligand that binds to a proteinpresent in those cells comprising the undesirable target but not presentin those cells that have the desired target. The particular presenterligand that is employed will necessarily depend on the nature of the twotypes of cells among which differentiation in drug activity is desired.For example, where the two types of cells are microbial and human cells,the presenter protein ligand may be one that binds to a protein that ispresent in the human cells but not present in the microbial cells, e.g.FKBPs, and the like. The bifunctional molecule/presenter protein binarycomplex is unable to bind to the undesirable targets in those cells thatdo not comprise the desired target. At the same time, the freebifunctional molecule is able to bind to the desired target in thosecells that comprise the desired target because of repulsive interactionsbetween the binary complex and the undesirable target. For example,where the drug moiety is a particular antibiotic that exhibits desiredactivity in bacterial cells but undesirable activity in human cells, thebifunctional molecule is able to bind to the bacterial target but thebifunctional molecule/binary complex is not able to bind to the humantarget(s). See FIG. 1C. As such, the bifunctional molecule provides forenhanced selectivity as compared to the free drug.

[0118] Preferred drugs in this third embodiment are molecules whichexhibit considerable side effects and toxicity in human cells inaddition to a desired activity in target cells. Many of these moleculestarget metabolic pathways and biological activities that are common tothe target cell as well as non-target cells, such as: inhibitors of RNApolymerase II like -aminitin, rifamycin, rifampicin and actinomycin D;protein synthesis inhibitors like cycloheximide, streptomycin,tetracycline, chloramphenicol, erythromycin or puromycin; dihydrofolatereductase inhibitiors like methotrexate; topoisomerase II inhibitorslike novobiocin and ciprofloxacin, proteasome inhibitors likelactacystin; channel inhibitors.

[0119] This embodiment finds particular use in the targeting of a drugto a microbial pathogen while reducing toxicity to the host in which themicrobial pathogen is present. Microbial pathogens that may be targetedinclude Legionella sp., Chlamydia sp., Staphylococcus sp., Neisseriasp., Rickettsia sp., Coxiella sp., Neurospora sp., Escherischia coli,Heliobacter pylori as well as protozoan pathogens like Plasmodium sp.,Leishmania sp., Trypanosoma sp., Entamoeba histolytica, Giardia lamblia,Trichomonas vaginalis.

[0120] In one particular embodiment of interest, the drug is selectedfrom the group consisting of methotrexate and puromycin which are linkedto a presenter ligand such that DHFR and protein synthesis,respectively, are less affected in cells containing the presenterprotein (e.g. FKBP), e.g. host cells, than in cells that lack thepresenter, e.g. pathogen cells.

[0121] Methods of use, Pharmaceutical Preparations and Kits

[0122] The subject bifunctional molecules find use in producing at leastbinary complexes in vivo, where the production of these complexes isdesirous to the host in which they occur, e.g. is beneficial to thehost. The term binary complex is used throughout this specification torefer to any complex produced by the non-covalent binding of twodistinct molecules, i.e. the bifunctional molecule and the presenterprotein or the bifunctional molecule and the protein target. In manypreferred embodiments, tripartite complexes are produced with thesubject bifunctional molecules. The term tripartite complex is usedthroughout this specification to refer to binding complexes of threedistinct entities, i.e. the protein drug target, the bifunctionalmolecule and the presenter protein.

[0123] In the methods of the subject invention, an effective amount ofthe bifunctional molecule is administered to the host, where “effectiveamount” means a dosage sufficient to produce the desired result, e.g. animprovement in a disease condition or the symptoms associated therewith.The bifunctional molecule may be administered to the host using anyconvenient means capable of producing the desired result. Thus, thebifunctional molecule can be incorporated into a variety of formulationsfor therapeutic administration. More particularly, the bifunctionalmolecule of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants and aerosols. As such, administration of thebifunctional molecule can be achieved in various ways, including oral,buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal,intracheal, etc., administration. In pharmaceutical dosage forms, thebifunctional molecule may be administered alone or in combination withother pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

[0124] For oral preparations, the bifunctional molecules can be usedalone or in combination with appropriate additives to make tablets,powders, granules or capsules, for example, with conventional additives,such as lactose, mannitol, corn starch or potato starch; with binders,such as crystalline cellulose, cellulose derivatives, acacia, cornstarch or gelatins; with disintegrators, such as corn starch, potatostarch or sodium carboxymethylcellulose; with lubricants, such as talcor magnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

[0125] The bifunctional molecules can be formulated into preparationsfor injection by dissolving, suspending or emulsifying them in anaqueous or nonaqueous solvent, such as vegetable or other similar oils,synthetic aliphatic acid glycerides, esters of higher aliphatic acids orpropylene glycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

[0126] The bifunctional molecules can be utilized in aerosol formulationto be administered via inhalation. The compounds of the presentinvention can be formulated into pressurized acceptable propellants suchas dichlorodifluoromethane, propane, nitrogen and the like.

[0127] Furthermore, the bifunctional molecules can be made intosuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. The compounds of the present invention canbe administered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

[0128] Unit dosage forms for oral or rectal administration such assyrups, elixirs, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of the composition containing activeagent. Similarly, unit dosage forms for injection or intravenousadministration may comprise the active agent in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

[0129] The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

[0130] The pharmaceutically acceptable excipients, such as vehicles,adjuvants, carriers or diluents, are readily available to the public.Moreover, pharmaceutically acceptable auxiliary substances, such as pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents and the like, are readily available to the public.

[0131] Those of skill will readily appreciate that dose levels can varyas a function of the specific compound, the severity of the symptoms andthe susceptibility of the subject to side effects. Preferred dosages fora given compound are readily determinable by those of skill in the artby a variety of means.

[0132] The subject methods find use in the treatment of a variety ofdifferent disease conditions. In certain embodiments, of particularinterest is the use of the subject methods in disease conditions wherean active agent or drug having desired activity has been previouslyidentified, but which active agent or drug does not bind to its targetwith desired affinity and/or specificity. With such active agents ordrugs, the subject methods can be used to enhance the binding affinityand/or specificity of the agent for its target.

[0133] The specific disease conditions treatable by with the subjectbifunctional compounds are as varied as the types of drug moieties thatcan be present in the bifunctional molecule. Thus, disease conditionsinclude cellular proliferative diseases, such as neoplastic diseases,autoimmune diseases, cardiovascular diseases, hormonal abnormalitydiseases, infectious diseases, and the like.

[0134] By treatment is meant at least an amelioration of the symptomsassociated with the disease condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thepathological condition being treated, such as inflammation and painassociated therewith. As such, treatment also includes situations wherethe pathological condition, or at least symptoms associated therewith,are completely inhibited, e.g. prevented from happening, or stopped,e.g. terminated, such that the host no longer suffers from thepathological condition, or at least the symptoms that characterize thepathological condition.

[0135] A variety of hosts are treatable according to the subjectmethods. Generally such hosts are “mammals” or “mammalian,” where theseterms are used broadly to describe organisms which are within the classmammalia, including the orders carnivore (e.g., dogs and cats), rodentia(e.g., mice, guinea pigs, and rats), and primates (e.g., humans,chimpanzees, and monkeys). In many embodiments, the hosts will behumans.

[0136] Kits with unit doses of the bifunctional molecule, usually inoral or injectable doses and often in a storage stable formulation, areprovided. In such kits, in addition to the containers containing theunit doses will be an informational package insert describing the useand attendant benefits of the drugs in treating pathological conditionof interest. Preferred compounds and unit doses are those describedherein above.

[0137] The following examples are offered by way of illustration and notby way of limitation.

EXPERIMENTAL EXAMPLE I Bifunctional Molecules of pYEEI

[0138] SH2 domains of the tyrosine kinases Fyn and Lck as well as theN-terminal SH2 domain of PLC were used to demonstrate that the affinityand specificity of a ligand that is bound by multiple proteins can beimproved through the use of presenter proteins. While Fyn, Lck and PLCyhave similar biochemical properties, they are involved in differentsignaling processes affecting different biological endpoints. The ligandstudied was a phosphotyrosine peptide with the sequence NH₂-pYEEI-COOH.This peptide binds to all three SH2 domains, Fyn, Lck and PLC [Songyanget al. Cell (1993) 72, 767].

[0139] A. Bifunctional Molecule Synthesis

[0140] 1. Synthesis of FKpYEEI

[0141] The pYEEI peptide was chemically linked to FK506 in order tocreate the bifunctional ligand, FKpYEEI (FIG. 2). The FK506 moiety canbe bound by the family of FK506 binding proteins, the FKBPs, which serveas the presenter proteins in the experiments described below. Thesynthesis of FKpYEEI is based on reacting an activated form of FK506, amixed carbonate, with the primary amino group of pYEEI. The preparationof the mixed carbonate involves several oxidation steps. To protect thetwo hydroxy groups from oxidation, a sample of FK506 was reacted withtert-butyldimethylsilyl triflate (TBSOTf) in the first reaction. A 10-mLvial was charged with FK506 (32 mg, 40 mol), 2,6-lutidine (47 L, 400mol, 10 eq), TBSOTf(47 L, 200 mol, 5 eq) and 3 mL of distilleddichloromethane. The reaction was stirred under an atmosphere ofnitrogen for 45 min, starting at 0° C. and letting it warm to roomtemperature. To quench any excess TBSOTf, 25 eq of methanol (40 L) wasadded and the reaction was stirred at room temperature for 25 minutes.The reaction was extracted twice with 10 mL DCM in the presence of 10 mLaqueous sodium bicarbonate. The organic phase, containing 24,32-bis[(tert-butyldimethylsilyl)oxy]-FK506 (FK506-TBS2), was dried withmagnesium sulfate and subsequently, filtered and evaporated to an oil.The product was purified by flash chromatography using a mixture ofhexane and ethyl acetate (5:1 to 3:1). The fractions containingFK506-TBS2 were combined and evaporated. The yield was 35 mg.

[0142] 2. Osmylation and Oxidative Cleavage

[0143] A 10 mL flask was charged with FK506-TBS2 (32 mg, 31 mol),4-methylmorpholine N-oxide (21 mg, 155 mol), water (50 L) andtetrahydrofuran (THF) (2 mL). Osmium tetroxide (26 L, 3.1 mol, 0.12 Msolution in water) was added via syringe. The clear colorless solutionwas stirred at room temperature for 4.0 h. The reaction was diluted with50% aqueous methanol (1 mL) and sodium periodate (66 mg) was added inone portion. The cloudy mixture was stirred 25 min at room temperature,diluted with ether (10 mL), and washed with a saturated aqueous sodiumbicarbonate solution (10 mL). The phases were separated and the aqueouslayer was back-extracted with ether (2×10 mL) The combined organiclayers were dried over MgSO4 and solid sodium sulfite (50 mg). Theorganic phase was then filtered and concentrated.

[0144] 3. Reduction

[0145] The aldehyde 1 was immediately dissolved in THF (2 mL) and cooledto −78° C. under an atmosphere of nitrogen, and treated with lithiumtris[(3-ethyl-3-pentyl)oxy]aluminum hydride (63 L, 8.8 mol, 1 eq). Theclear solution was stirred for 60 min at −78° C., then quenched withether (3 mL) and saturated aqueous ammonium chloride (200 L). Themixture was allowed to warm to room temperature and solid sodium sulfatewas added to dry the solution. The mixture was stirred 20 min, filtered,concentrated, and the resulting oil was immediately dissolved inacetonitrile (2 mL).

[0146] 4. Mixed Carbonate

[0147] To the solution of the primary alcohol 2 in acetonitrile (2 mL)was added 2,6-lutidine (36 1, 0.31 mmol, 10 eq) and N,N′-disuccinimidylcarbonate (40 mg, 0.16 mmol, 5 eq). The heterogenous mixture was stirredat room temperature for 19 h, at which time the solution was dilutedwith ether (10 mL) and washed with saturated aqueous sodium bicarbonate(5 mL). The aqueous solution was back-extracted with ether (2×10 mL).The organic phases were combined and dried (MgSO4), concentrated andsubjected to flash chromatography (3:1 to 2:1 to 1:1 hexane/ethylacetate). The desired mixed carbonate 3 was isolated as a clear,colorless oil (7.1 mg, 6 mmol).

[0148] 5. Coupling of the pYEEI peptide

[0149] A 1 mL-vial was loaded with the mixed carbonate (7 mg, 5.95 mol),pYEEI peptide (7.7 mg. 11.9 mol, 2 eq) and triethylamine (21 L, 119 mol,20 eq) together with dimethylformamide (400 L). The reaction was stirredat room temperature for 24 h. For the deprotection of TBS2-FKpYEEI, thereaction was transfered into a 1.5 mL polypropylene eppendorf tube.Acetonitrile (100 L) was added to bring the reaction volume to 500 L.Then, hydrofluoric acid (55 L) was pipeted into the reaction. Thereaction was stirred for 16 h and, then, quenched with triethylamine(160 L). The reaction mixture was lyophilized to a yellow oil. The oilwas taken up in 33% aqueous acetonitrile and 0.1% trifluoroacetic acid.The separation of FKpYEEI from unreacted peptide was achieved by HPLC(column: Supelco LC-18 C18, 100 Å). By amino acid analysis it was foundthat the yield of the synthesis was 1 mg of FKpYEEI.

[0150] B. Synthesis of SLFpYEEI

[0151] SLF was synthesized according to the procedures of Holt et al.[J. Am. Chem. Soc. 1993, 115, 9925]. SLF was coupled to the N-terminusof the resin-bound protected pYEEI peptide using PyBOP. Theheterodimeric SLFpYEEI was deprotected and cleaved from the Novasyn TGTresin (Calbiochem-Novabiochem, San Diego, Ca.) using 25% trifluoroaceticacid and 2.5% triisopropyl silane in methylene chloride and the desiredproduct was isolated using reverse phase HPLC. C. Recombinant FKBPs Aspresenter proteins, we used FKBP12 and FKBP52, two FKBPs that bind FK506with high affinity (0.4 nM and 44 nM, respectively). Recombinant FKBP12or FKBP52 bind to the FK506 moiety of FKpYEEI, thus creating an enlargedbinding surface. Recombinant FKBP12 was expressed as a GST fusionprotein and bound to glutathione beads (Pharmacia). Using thrombin(Sigma) to cleave the GST-FKBP12 fusion protein at a thrombin cleavagesite in the linker between GST and FKBP12, recombinant FKBP12 could bereleased from the glutathione beads where GST remained bound. FKBP52 wasexpressed with a tag of six histidine residues at its N-terminus (pET28cexpression vector, Novagen). The recombinant protein was bound via thehistidine tag to Ni²⁺ NTA agarose beads (Qiagen), washed extensively andeluted with imidazole. The FKBP52 elution buffer was dialyzed over nightagainst the buffer used in the binding reactions (20 mM Tris pH 7.2, 150mM NaCl). In order to establish the affinity of the FKBP-FKpYEEI complexfor the three SH2 domains, the following competition assay wasdeveloped.

[0152] D. The Assay

[0153] The pYEEI peptide was coupled to Affi-Gel 10 beads (Biorad) viaits amino-group. The Fyn, Lck and PLC SH2 domains were expressed inbacteria as GST fusion proteins. The linker between the GST protein andthe SH2 domain contained a thrombin cleavage site and a protein kinase A(PKA) phosphorylation site. The fusion protein on glutathione beads wasradioactively labeled at the PKA phosphorylation site using PKA (Sigma).After extensive washing of the beads, the radioactive SH2 domain wasreleased by cleavage with thrombin (Sigma). In a typical binding assay,7.5 L of a 1:1 peptide bead slurry were incubated with the radioactiveSH2 domain (200 nM) for 2 hours in 100 L of binding buffer (20 mM TrispH7.2; 150 mM NaCl). In order to separate the radioactive protein boundto the beads, the binding reaction was transfered into a PCR tube (USAScientific Plastic), whose bottom had been punctured with a 26gaugeneedle. The PCR tube was placed in a 0.5 mL Eppendorf tube which in turnwas placed in a regular 1.5 mL Eppendorf tube. The tubes were spun for 1second at maximal speed in an Eppendorf centrifuge resulting in theseparation of the beads remaining in the PCR tube and the supernatant,which was collected in the 0.5 ml Eppendorf tube. The beads wereresuspended in 100 L PBS and the radioactivity was counted in a liquidscintillation counter after addition of 3 ml scintillation fluid. Theradioactivity remaining in the 100 L supernatant was counted in the sameway. It was determined that under the conditions described 65% Fyn, 25%PLC and 15%Lck SH2 domain were bound to the beads. By increasing theamount of beads, up to 80% Fyn, 40% PLC and 25% Lck could be bound.These amounts reflect the active fraction of SH2 domains in thedifferent protein preparations. There was no indication that theinactive protein fraction interfered with the binding assay.

[0154] 1. Experiments:

[0155] SLFpYEEI and FKpYEEI are bifunctional molecules that can bindFKBP12 and FKBP52 as well as the Fyn SH2 domain (FIG. 2). The bindingconstants (Kd values) of the bifunctional molecules for the threeproteins were measured using isothermal titration calorimetry (ITC)(Table 1). TABLE 1 Binding Constants (Kds) for Interacting Partners Usedin this Study FKpYEEI SLFpYEEI SLF FK506 FKBP12  45 nM  61 nM 20 nM 0.4nM FKBP52 150 nM  5 M  3 M 44-66 nM Fyn SH2 520 nM 183 nM n.d. n.d.domain # The syringe was loaded into the cell, spun at 400 rpm andallowed to equilibrate for 1-2 hr. Ligand was injected into the cell (25× 10 L, 6 min apart) and the heat evolved was quantitated. Bindingconstants were calculated from a numerical fit to the experimental dataas described in (Wiseman et al., Anal. Biochem. (1989), 179, 131) .

[0156] In order to determine the relative affinity of the SH2 domainsfor FKpYEEI or SLFpYEEI alone and FKpYEEI or SLFpYEEI bound to FKBP12and FKBP52, the following competition assay was used. Radioactive SH2domains were incubated with pYEEI peptide beads and FKpYEEI or SLFpYEEIin the presence or absence of the respective FKBP proteins. FKpYEEI andFKpYEEI/FKBP as well as SLFpYEEI and SLFpYEEI/FKBP compete for bindingof the SH2 domains with the peptide beads. The higher the affinity ofthe FKpYEEI or SLFpYEEI ligand in the supernatant, the less SH2 domainis bound to the peptide beads (FIG. 3).

[0157] The following experiments suggest that the formation of a binarycomplex between an endogenous protein like FKBP and a bifunctionalmolecule like FKpYEEI or SLFpYEEI can enhance the affinity, thespecificity and selectivity of a small molecule ligand such as the pYEEIpeptide.

[0158] a. Affinity Enhancement

[0159] i. In the presence of FKBP52, FKpYEEI can Bind the Fyn SH2 Domainwith Higher Affinity.

[0160] Using the competition binding assay described above, FKpYEEI wasadded to peptide beads and the Fyn SH2 domain in the presence ofincreasing concentrations of FKBP52 (FIG. 4). In this assay, in 100 Lbinding reactions, radioactively labeled Fyn SH2 domain (200 nM) wasincubated with 7.5 L of a 1:1 pYEEI bead slurry. No FKpYEEI, 1.0 MFKpYEEI or 1.0 M FKpYEEI plus increasing concentrations of FKBP52(0.25-4.0 M) were added. The binding reactions were rotated at roomtemperature for 2 hours. The supernatant was separated from the beads byspinning the binding reactions in punctured PCR tubes for 1 sec in anEppendorf centrifuge at maximal speed. The beads were resuspended in 1001 PBS and the radioactivity bound to the beads was counted in a liquidscintillation vial for 30 sec. The radioactivity bound to the beads inthe presence of only the Fyn SH2 domain was plotted as 100% of maximalFyn SH2 domain binding to pYEEI beads. The radioactivity bound in thepresence of FKpYEEI and FKBP52 was calculated as the percentage ofmaximal Fyn SH2 domain binding. Every binding reaction was done intriplicate. The data points reflect the average of the three bindingreactions. The error bars indicate the standard error.

[0161] The results show that as more FKBP52 is present, less Fyn isbound by the peptide beads. FKpYEEI binds to FKBP52 and forms a binarycomplex. In the context of this binary complex, FKpYEEI has a higheraffinity for the Fyn SH2 domain which is reflected in the decreasingamounts of Fyn SH2 domain binding to peptide beads.

[0162] ii. FK506 Reverses the FKBP52 Effect

[0163] To verify that the affinity enhancing effect is based on a binarycomplex formed by FKpYEEI binding to the FK506 binding pocket of FKBP52,increasing concentrations of FK506 were added to the binding reactioncontaining the Fyn SH2 domain, FKpYEEI and FKBP52 (FIG. 5). In thisassay, in 100 L binding reactions, radioactively labeled Fyn SH2 domain(200 nM) was incubated with 7.5 L of a 1:1 pYEEI bead slurry. FKpYEEI(1.0 M), FKpYEEI (1.0 M) plus FKBP52 (2.0 M) and FKpYEEI (1.0 M) plusFKBP52 (2.0 M) plus increasing concentrations of FK506 (0.25-15.0 M)were added. The binding reactions were rotated at room temperature for 2hours, centrifuged and analysed as described above.

[0164] The results show that as the concentration of FK506 increases,the affinity enhancing effect of FKBP52 is reversed. Free FK506 binds tothe FK506 binding pocket of FKBP52 with higher affinity than FKpYEEI(see Table 1). Therefore, the loss of the affinity enhancing effect isdue to the dissociation of the binary complex formed between FKBP52 andFKpYEEI.

[0165] iii. The Presence of FKBP52 Increases the Affinity of FKpYEEI forthe Fyn SH2 Domain by Three-Fold

[0166]FIG. 6 is a graph of the competition binding curves for the FynSH2 domain and FKpYEEI as well as the Fyn SH2 domain and the peptidepYEEI in the absence and the presence of FKBP52. Fyn SH2 domain wasincubated with peptide beads and increasing concentrations of FKpYEEI inthe absence or presence of FKBP52. Specifically, in this assay, in 100 Lbinding reactions, radioactively labeled Fyn SH2 domain (200 nM) wasincubated with 7.5 L of a 1:1 pYEEI bead slurry. Increasingconcentrations of FKpYEEI or pYEEI (0.1-20.0 M) were added in thepresence or absence of FKBP52 (4.5 M). The binding reactions wererotated at room temperature for 2 hours, centrifuged and analysed asdescribed above. Half maximal binding (IC50) is observed at 750 riMFKpYEEI in the absence of FKBP52. In the presence of FKBP52, the IC50value is 250 riM. Hence, in a complex with FKBP52, the affinity ofFKpYEEI for the Fyn SH2 domain is enhanced by a factor of three. TheIC50 of the peptide pYEEI is 1.1 M. Hence, FKpYEEI in the context ofFKBP52 shows a 4.4 fold enhancement of affinity in respect to pYEEI.Using ITC, the binding constant (Kd) for Fyn SH2 domain binding to theFKpYEEI-FKBP52 complex was determined. The Kd value for this bindingevent is 130 nM. This is a fourfold enhancement over the Kd of Fyn SH2domain binding to FKpYEEI alone (Table 1) and this result confirms thedata obtained from the competition binding assay.

[0167] iv. The Presence of FKBP12 does not Increase the Affinity ofFKpYEEI for the Fyn SH2 Domain.

[0168] The structure of the FK506 binding domain of FKBP52 is verysimilar to the structure of FKBP12 (Craescu et al., Biochemistry 1996,35, 11045). To test if the observed affinity enhancement can be achievedby the formation of a binary complex with FKBP12, increasingconcentrations of FKBP12 were added to binding reactions containing theFyn SH2 domain and FKpYEEI (FIG. 7). In this assay, in 100 L bindingreactions, radioactively labeled Fyn SH2 domain (200 nM) was incubatedwith 7.5 L of a 1:1 pYEEI bead slurry. No FKpYEEI, 1.0 M FKpYEEI or 1.0M FKpYEEI plus increasing concentrations ofFKBP12 (0.25-4.0 M) wereadded. The binding reactions were rotated at room temperature for 2hours, centrifuged and analysed as described above. The results indicatethat the formation of a binary complex between FKpYEEI and FKBP12 doesnot enhance the binding affinity for the Fyn SH2 domain.

[0169] In summary, the above experiments show that a binary complex ofFKBP52 and FKpYEEI binds to the Fyn SH2 domain with a higher affinitythan FKpYEEI or pYEEI alone. Since FKBP12 binds FKpYEEI in the same wayas FKBP52 but does not support this effect, the increase in affinitymust be based on favorable protein-protein interactions between the FynSH2 domain and FKBP52.

[0170] b. Specificity Enhancement

[0171] i. FKBP12 Reduces the Affinity of FKpYEEI for the PLC SH2 Domain

[0172] The binary complex of FKpYEEI and FKBP 12 has the same affinityfor the Fyn SH2 domain as free FKpYEEI (see FIG. 7). To test if theFKpYEEI-FKBP12 complex has an effect on the binding of the PLC SH2domain, increasing concentrations of FKBP12 were added to bindingreactions containing the PLC SH2 domain and FKpYEEI (FIG. 8). In thisassay, in 100 L binding reactions, radioactively labeled PLC SH2 domain(400 nM) was incubated with 7.5 L of a 1:1 pYEEI bead slurry. NoFKpYEEI, 1.0 M FKpYEEI or 1.0 M FKpYEEI plus increasing concentrationsof FKBP12 (0.5-4.0 M) were added. The binding reactions were rotated atroom temperature for 2 hours, centrifuged and analysed as describedabove. The results show that as the amount ofFKBP12 increases, more PLCSH2 domain binds to the peptide beads. Hence, the binary complex ofFKpYEEI-FKBP12 has a decreased affinity for the PLC SH2 domain ascompared to free FKpYEEI.

[0173] ii. The Reduction in Affinity of FKpYEEI for the PLC SH2 Domainin the Presence of FKBP12 is Reversed by FK506.

[0174] To test if the reduction of affinity is due to FKpYEEI binding inthe FK506 binding pocket of FKBP12, increasing concentrations of FK506were added to binding reactions containing the PLC SH2 domain, FKpYEEIand FKBP12 (FIG. 9). In this assay, in 100 L binding reactions,radioactively labeled PLC SH2 domain (400 nM) was incubated with 7.5 Lof a 1:1 pYEEI bead slurry. FKpYEEI (1.5 M), FKpYEEI (1.5 M) plus FKBP12(2.0 M) and FKpYEEI (1.5 M) plus FKBP12 (2.0 M) plus increasingconcentrations of FK506 (1.0-5.0 M) were added. The binding reactionswere rotated at room temperature for 2 hours, centrifuged and analysedas described above. The results show that as the concentration of FK506increases, the affinity reduction is reversed. Hence, the loss ofaffinity of FKpYEEI for the PLC SH2 domain is dependent on the formationof the binary complex with FKBP12. This suggests that unfavorableprotein-protein contacts between FKBP12 and the PLC SH2 domain are thebasis for the reduced binding.

[0175] iii. The Binary FKBP 12-FKpYEEI Complex Reduces the Affinity ofFKpYEEI for the PLC SH2 Domain but not for the Fyn or Lck SH2 Domain.

[0176] To test if the presence of FKBP12 affects the affinity of FKpYEEIfor other SH2 domains aside from the PLC SH2 domain, FKpYEEI and FKBP12were added to binding reactions containing the Fyn, Lck or PLC SH2domains (FIG. 10). In this assay, 100 L binding reactions, radioactivelylabeled PLC SH2 domain (200 iM), Lck SH2 domain (800 riM) and Fyn SH2domain (200 nM) was incubated with 7.5 L of a 1:1 pYEEI bead slurry.FKpYEEI (1.5 M), FKpYEEI (1.5 M) plus FKBP12 (2.0 M) and FKpYEEI (1.5 M)plus FKBP12 (2.0 M) plus FK506 (4.0 M) were added to the PLC SH2 domainreactions. FKpYEEI (1.0 M), FKpYEEI (1.0 M) plus FKBP12 (2.0 M) andFKpYEEI (1.0 M) plus FKBP12 (2.0 M) plus FK506 (4.0 M) were added to theLck and Fyn SH2 domains. The binding reactions were rotated at roomtemperature for 2 hours, centrifuged and analysed as described above.The results show that the FKpYEEI-FKBP12 complex does not affect theaffinity of FKpYEEI for the Fyn or Lck SH2 domain. Hence, theFKpYEEI-FKBP12 complex creates specificity by supporting the binding ofthe Fyn and Lck SH2 domains while reducing binding to the PLC SH2domain.

[0177] In summary, the above experiments show that the formation of abinary complex may lead to unfavorable protein-protein interactionsbetween the presenter protein and some targets but not other targets ofthe drug. Therefore, the formation of a complex between a bifunctionalmolecule and a presenter protein can be used to create specificity.

[0178] c. Selectivity Enhancement

[0179] i. FKBP 12 Reduces the Affinity of SLFPYEEI for the Fyn SH2Domain.

[0180] In comparison to FKpYEEI, SLFpYEEI presents the pYEEI peptide ina different orientation and in a different distance in respect to theFKBP12 protein surface. To test if the binary SLFpYEEI-FKBP12 complexaffects binding of the Fyn SH2 domain differently than free SLFpYEEI,increasing concentrations of FKBP12 were added to binding reactionscontaining SLFpYEEI and the Fyn SH2 domain (FIG. 11). In this assay, in100 L binding reactions, radioactively labeled Fyn SH2 domain (200 nM)was incubated with 7.5 L of a 1:1 pYEEI bead slurry. SLFpYEEI (1.0 M)and SLFpYEEI (1.0 M) plus increasing concentrations of FKBP12 (0.25-4.0M) were added. The binding reactions were rotated at room temperaturefor 2 hours, centrifuged and analysed as described above. The additionof FKBP12 increases the amount of Fyn SH2 domain binding to peptidebeads. This indicates that the SLFpYEEI-FKBP12 complex shows reducedaffinity for the Fyn SH2 domain.

[0181] ii. FK506 Reverses the Effect of Decreased Binding Activity ofthe SLFpYEEI-FKBP12 Complex.

[0182] To verify that the reduced affinity of the Fyn SH2 domain isbased on SLFpYEEI binding to the FK506 binding pocket of FKBP12,increasing concentrations of FK506 were added to binding reactionscontaining Fyn SH2 domain, SLFpYEEI and FKBP12 (FIG. 12).In this assay,in 100 L binding reactions, radioactively labeled Fyn SH2 domain (200nM) was incubated with 7.5 L of a 1:1 pYEEI bead slurry. SLFpYEEI (1.0M) and SLFpYEEI (1.0 M) plus FKBP12 (2.0 M) and SLFpYEEI (1.0 M) plusFKBP12 plus increasing concentrations of FK506 (1.0-3.0 M) were added.The binding reactions were rotated at room temperature for 2 hours,centrifuged and analysed as described above. FK506 reverses the effectof reduced affinity of SLFpYEEI for the Fyn SH2 domain in the presenceof FKBP12. Hence, the binary complex of SLFpYEEI and FKBP 12 establishesunfavorable protein-protein interactions between FKBP 12 and the Fyn SH2domain so that the affinity, with which the binding event is takingplace, is reduced.

[0183] iii. The Presence of FKBP12 Decreases the Affinity of SLFpYEEIfor the Fyn SH2 Domain by Six-Fold

[0184]FIG. 13 is a graph of the competition binding curves for the FynSH2 domain and SLFpYEEI in the absence and the presence of FKBP12. FynSH2 domain was incubated with increasing concentrations of SLFpYEEI inthe absence or presence of FKBP12. In this assay, in 100 L bindingreactions, radioactively labeled Fyn SH2 domain (200 nM) was incubatedwith 7.5 L of a 1:1 pYEEI bead slurry. Increasing concentrations ofSLFpYEEI (0.1-20.0 M) were added in the presence or absence of FKBP12(20 M). The binding reactions were rotated at room temperature for 2hours, centrifuged and analysed as described above. Half maximal binding(IC50) is observed at 0.25 M SLFpYEEI in the absence ofFKBP12. In thepresence ofFKBP12, the IC50 value is 1.5 M. Hence, in a complex withFKBP12, the affinity of SLFpYEEI for the Fyn SH2 domain is reduced by afactor of six. This effect was confirmed by ITC. The Kd of Fyn SH2domain binding to free SLFpYEEI is 183 nM (Table 1) and the Kd forbinding of the SLFpYEEI-FKBP12 complex is 1.5 M.

[0185] iv. The SLF YEEI-FKBP12 Complex Shows Reduced Binding to the Fvnas well as the Lck and PLC SH2 Domains.

[0186] To test if the PLC and the Lck SH2 domains bind toSLFpYEEI-FKBP12 with reduced affinity, these SH2 domains together withthe Fyn SH2 domain were incubated with SLFpYEEI alone or SLFpYEEI andFKBP12 (FIG. 14). In this assay, in 100 L binding reactions,radioactively labeled Fyn, Lck and PLC SH2 domains (200 nM) wereincubated with 7.5 L of a 1:1 pYEEI bead slurry. SLFpYEEI (1.0 M) andSLFpYEEI (1.0 M) plus FKBP12 (2.0 M) and SLFpYEEI (1.0 M) plus FKBP12(2.0 M) plus FK506 (3.0 M) were added. The binding reactions wererotated at room temperature for 2 hours, centrifuged and analysed asdescribed above. Not only the Fyn SH2 domain, but also the Lck and PLCSH2 domains show reduced binding by the binary complex.

[0187] In summary, the above experiments show that the formation of abinary complex may greatly reduce binding of the drug to all of itstargets in a cell that contains the presenter molecule. If an organismhas cells that contain the presenter protein and other cells that do notcontain the presenter, then, the cells lacking the presenter proteinwill be more affected by the activity of the bifunctional molecule thancells expressing the presenter.

EXAMPLE II Activity of Penicillamine Para-Amino Salicylic AcidBifunctional Molecule

[0188] To determine whether isoform specificity could be achieved bychemically linking a drug to a ligand for an extracellular protein, wesynthesized compound 1 shown below in FIG. 1. Compound 1 consists of apenicillamine moiety coupled via a glycine linker to para-aminosalicylic acid, a known ligand for albumin with an IC50 of 5 μm.Penicillamine is an inhibitor of the enzyme alkaline phosphatase, withIC 50 values in the μM range for a wide variety of alkaline phosphataseisoforms. We reasoned that by recruiting albumin to the alkalinephosphatase surface using the bifunctional compound 1, we could modulatethe effective affinity of penicillamine for its target, alkalinephosphatase.

[0189] Reactions were conducted in 200 mM diethanolamine buffer, pH 10,with methylumbeliferal phosphate as the substrate. The reaction wasmonitored by recording fluorescence of the hydrolysis product(excitation at 355 nM, fluorescence at 460 nM), and the first 10% of thereaction was used to determine the rate of the reaction.

[0190] The results are tabulated in table 2 below. In the presence of100 μM human serum albumin, compound 1 was seen to be a better inhibitorfor four of the enzymes tested relative to the inhibition seen in theabsence of albumin. For the other eight enzymes tested, no change in theIC50 value was seen in the presence or absence of albumin. IC50 valueswere seen to decrease roughly 2-fold for the eel intestine and humanplacenta isoforms of the enzyme, and roughly 12-fold for the rabbitintestine and shrimp isoforms. TABLE 2 IC50 Values of Compound 1 forAlkaline Phosphatase Isoforms in the Presence and Absence of 100 μMHuman Serum Albumin IC50 Enzyme Source IC50 (no albumin) (100 μMalbumin) Calf Intestine 20 μM 20 μM Rabbit Intestine 300 μM  25 μMBacteria 50 μM 50 μM Dog Intestine 20 μM 20 μM Porcine Kidney 50 μM 50μM Guinea Pig Intestine 80 μM 80 μM Eel Intestine 110 μM  70 μM PorcineIntestinal Mucosa 50 μM 50 μM Bovine Milk 50 μM 50 μM Human Placenta 300μM  45 μM Shrimp  3 mM 80 μM

EXAMPLE III Selectivity Experiment-Using bifunctional Molecules toDetoxify Antimicrobials

[0191] A. Introduction

[0192] Many molecules that contain the 2,4-diaminopteridine bicyclicring structure are inhibitors of dihydrofolate reductase (DHFR) andthese molecules usually possess strong antimicrobial activity. However,the pteridine structure alone is not very attractive for widespread useas an anti-infective because it is a non-selective inhibitor of DHFR: itaffects not only microbial DHFR but it also inhibits the human homolog.As a result, treatment of a patient with pteridines often causes sideeffects and toxicity. In order to reduce the ability of the pteridinenucleus to bind to human DHFR, a pteridine derivative is covalentlylinked to a ligand of FKBP (e.g. SLF, FK506, etc.). Based onstructure-activity relationship (SAR) data, the pteridine and FKBPligand are linked in such a way that their binding to DHFR and FKBP,respectively, are as little affected as possible.

[0193] B. Synthesis

[0194] The following provides a representative synthesis protocol forthe production of a pteridine-SLF bifunctional compound. Analogousprotocols are employed to produce bifunctional compounds of pteridineand other FKBP ligands, such as FK506.

[0195] In the case of the pteridines, SAR data suggests that the alkylposition at C6 is the best for derivatization in order not to disruptbinding to DHFR. The co-crystal structure of SLF and FKBP together withSAR data reveals that the best position to derivative SLF is thephenolic hydroxyl group. To obtain a molecule with the desiredproperties, various linkers are used to connect the pteridine and SLF.Two examples are shown.

[0196] The synthesis of two pteridine-SLF bifunctional molecules withtwo different linkers requires the following reactions:

EXAMPLE 1

[0197] A solution of 2,4-diamino-6-bromomethylpteridine (1) indimethylacetamide (Rosowsky et al., J. Med. Chem., 1985, 28, 660-667) iscombined with synthetic FKBP ligand 2 (D. Holt et al., JACS, 1993, 115,9925) with 2 equivalents of potassium tert-butoxide and stirred at roomtemperature for 24-48 h. The reaction is monitored by thin layerchromatography (TLC, 9/1 chloroform/methanol). When the reaction iscomplete, the solvent is removed under reduced pressure, and the productis purified using silica gel chromatography with 19/1chloroform/methanol as eluent to provide the desired bifunctionalmolecule 3.

EXAMPLE 2

[0198] A solution of 2,4-diamino-6-bromomethylpteridine (1) (Rosowsky,et al.) is dissolved in dimethylacetamide and combined with 1 equivalentof carbobenzoxyhydrazide and 1 equivalent of barium oxide and stirredunder an argon atmosphere at 46° C. for 24 h. The reaction is monitoredby TLC with 9/1 chloroform/methanol and purified directly using silicagel chromatography with 9/1 chloroform/methanol as the eluent. Theintermediate product is reduced using catalytic hydrogenation with 10%Pd/C catalyst and atmospheric hydrogen pressure in methanol. Thereaction is monitored by TLC (4/1 chloroform/methanol) and product 4 isisolated by filtering the mixture through celite to remove the solidcatalyst and removing methanol under reduced pressure. Product 4 iscoupled to SLF (5) (Holt et al) using dicyclohexylcarbodiimide andcatalytic DMAP in dimethylformamide. The reaction is monitored by TLC(19/1 chloroform/methanol) and product 6 is isolated using silica gelchromatography (19/1 chloroform/methanol as eluent).

[0199] C. Assays

[0200] In vitro Assay

[0201] The resulting bifunctional pteridine-FKBP ligand molecules aretested in an in vitro DHFR inhibition assay to select a molecule withthe desired properties. The desired molecule fulfills the followingrequirements:

[0202] 1. In a DHFR inhibition assay the desired pteridine-FKBP ligandbifunctional molecule inhibits the acitivity of DHFR in a concentrationdependent manner. Molecules that are not able to inhibit DHFRefficiently are discarded. In these molecules, the linker between thepteridine and FKBP ligand affects the activity of the pteridine.

[0203] 2. When the in vitro DHFR inhibiton assay is repeated in thepresence of increasing concentrations of FKBP, the ability of thedesired pteridine-FKBP ligand molecule to inhibit DHFR is decreased incorrelation to increasing FKBP concentrations. Pteridine-FKBP ligandmolecules, which can inhibit DHFR despite FKBP, are discarded becausethe linkers of these molecules interfere with bifunctional moleculebinding to FKBP or the linkers do not support steric clashes between theFKBP and DHFR surfaces.

[0204] 3. The activity reduction of the desired pteridine-FKBP ligandbifunctional molecule in the presence of FKBP is based on the binding toFKBP via the FKBP ligand moiety. To demonstrate this, the assay isrepeated in the presence of pteridine-FKBP ligand bifunctional molecule,FKBP and increasing concentrations of FK506 which competes with the FKBPligand moiety of bifunctional molecule for FKBP binding. As theconcentration of FK506 is increased, the activity of pteridine-FKBPligand bifunctional molecule is regained because FK506 replaces theligand moiety as the ligand for FKBP. The free pteridine-FKBP ligandbifunctional molecule can again bind and inhibit the activity of DHFR.

[0205] The desired bifunctional molecule fulfills the three statedrequirements. Its use as a selective inhibitor is demonstrated in thefollowing cell based assays.

[0206] Bacterial Assay

[0207] The desired bifunctional molecule can inhibit DHFR activity invivo. E. coli does not express FKBP and the cells have no other FKBPligand (e.g. SLF or FK506) binding protein. When E. coli cultures areincubated with increasing concentrations of bifunctional molecule, thegrowth of the cultures is inhibited in a concentration dependentfashion. When these cells are transformed with a vector that allows theinducible expression of human FKBP, the bacteria become less sensitiveto the bifunctional molecule when FKBP is expressed. To demonstrate thatthis detoxification of the bifunctional molecule is based on binding toFKBP, increasing concentrations of FK506 are added to the culturemedium. FK506 competes with the bifunctional molecule for FKBP bindingso that increasing concentrations of free bifunctional moelcule arepresent which can inhibit DHFR. FK506 by itself has no effect on thegrowth of the bacterial cultures in the presence or absence of FKBP.

[0208] This assay demonstrates that the presence of FKBP protects cellsfrom the DHFR inhibitory activity of pteridine-FKBP ligand bifunctionalmolecules. This observation is the basis for creating cell selectiveanti-microbials that show reduced toxicity in humans.

[0209] It is evident from the above results and discussion that thesubject invention provides a powerful tool for improving the affinityand/or specificity and selectivity of drugs. As such, the subject methodprovides for the improvement of drugs currently in use, e.g. by reducingunwanted side effects. Furthermore, the subject methods can be used toimprove drugs that have, until now, been clinically useless due toconsiderable toxicity in humans and animals. Therefore, the inventionprovides for the potential usefulness of the variety of previouslydiscovered and discarded biologically active compounds. Accordingly, theinvention provides an important advancement in pharmacological science.

[0210] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. The citation of anypublication is for its disclosure prior to the filing date and shouldnot be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.

[0211] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A non-naturally occurring bifunctional moleculeof less than about 5000 daltons consisting of a drug moiety and apresenter protein ligand, wherein said drug moiety and said presenterprotein ligand are optionally joined by a linking group and said drugmoiety has enhanced activity as compared to a free drug control.
 2. Thebifunctional molecule according to claim 1, wherein said bifunctionalmolecule comprises a linking group.
 3. The bifunctional moleculeaccording to claim 1, wherein said drug moiety exhibits at least one ofenhanced affinity, specificity or selectivity for its target as comparedto a free drug control.
 4. The bifunctional molecule according to claim1, wherein said drug moiety binds to a protein target.
 5. Thebifunctional molecule according to claim 1, wherein said presenterprotein ligand binds to an extracellular protein.
 6. The bifunctionalmolecule according to claim 1, wherein said presenter protein ligandbinds to an intracellular protein.
 7. The bifunctional moleculeaccording to claim 6, wherein said presenter protein ligand is a ligandfor a peptidyl prolyl isomerase.
 8. A synthetic bifunctional molecule ofless than about 5000 daltons of the formula: Z—L—X wherein: X is a drugmoiety; L is a bond or a linking group; and Z is a ligand for anendogenous presenter protein ligand; wherein X and Z are different andsaid drug moiety has enhanced activity as compared to a free drugcontrol.
 9. The bifunctional molecule according to claim 8, wherein saiddrug moiety exhibits at least one of enhanced affinity, specificity orselectivity for its target as compared to a free drug control.
 10. Thebifunctional molecule according to claim 8, wherein said drug moiety hasa molecular weight of from about 50 to 2000 D.
 11. The bifunctionalmolecule according to claim 8, wherein said drug moiety binds to aprotein target.
 12. The bifunctional molecule according to claim 8,wherein said presenter protein ligand binds to an extracellular protein.13. The bifunctional molecule according to claim 8, wherein saidpresenter protein ligand binds to an intracellular protein.
 14. Thebifunctional molecule according to claim 13, wherein said presenterprotein ligand is a ligand for a peptidyl prolyl isomerase.
 15. Thebifunctional molecule according to claim 8, wherein said presenterprotein ligand has substantially no pharmacologic activity apart frombinding to a presenter protein ligand.
 16. A method for producing abinary complex in a host, said method comprising: administering to saidhost an effective amount of a bifunctional molecule of less than about5000 daltons consisting of a drug moiety and a ligand for a presenterprotein endogenous to said host, wherein said drug moiety and ligand areoptionally joined by a linking group; whereby a binary complexcomprising said bifunctional molecule and presenter protein is producedthat exhibits enhanced drug activity as compared to a free drug control.17. The method according to claim 16, wherein said enhanced drugactivity comprises at least one of enhanced affinity, specificity orselectivity of said drug moiety for a target of said drug moiety. 18.The method according to claim 16, wherein said drug moiety binds to aprotein target.
 19. The method according to claim 16, wherein saidpresenter protein endogenous to said host is naturally present at leastin the region of said target.
 20. The method according to claim 16,wherein a tripartite complex is produced between said bifunctionalmolecule, presenter protein and a target of said drug moiety, whereinsaid tripartite complex is characterized by the presence of presenterprotein target binding interactions.
 21. The method according to claim20, wherein said tripartite complex is produced intracellularly.
 22. Themethod according to claim 20, wherein said tripartite complex isproduced extracellularly.
 23. A method for producing a tripartitecomplex in a mammalian host, said method comprising: administering tosaid mammalian host an effective amount of a bifunctional molecule ofless than about 5000 daltons consisting of a drug moiety and a ligandfor a presenter protein endogenous to said mammalian host, wherein saiddrug moiety and ligand are optionally joined by a linking group; wherebysaid bifunctional molecule binds to a target of said drug and saidpresenter protein to produce said tripartite complex in said mammalianhost, wherein said tripartite complex is characterized by the presenceof presenter protein target binding interactions which result inenhanced drug activity as compared to a free drug control.
 24. Themethod according to claim 23, wherein said tripartite complex isproduced intracellularly.
 25. The method according to claim 23, whereinsaid tripartite complex is produced extracellularly.
 26. The methodaccording to claim 23, wherein said drug target is a protein.
 27. Themet hod according to claim 23, wherein said endogenous presenter proteinis selected from the group consisting of: peptidyl-prolyl isomerases,Hsp90, steroid hormone receptors, cytoskeletal proteins, albumin andvitamin receptors.
 28. The method according to claim 23, wherein saidbifunctional molecule is administered as a pharmaceutical preparation.29. A method for producing an intracellular tripartite complex in amammalian host, said method comprising: administering to said mammalianhost an effective amount of a bifunctional molecule comprising a drugmoiety and an endogenous presenter protein ligand, wherein the target ofsaid drug and said endogenous presenter protein are intracellularproteins; whereby said bifunctional molecule binds to said drug targetand endogenous presenter protein to intracellularly produce saidtripartite complex, wherein said tripartite complex is characterized bythe presence of presenter protein target binding interactions whichresult in enhanced drug activity as compared to a free drug control. 30.The method according to claim 29, wherein said target protein is anenzyme
 31. The method according to claim 29, wherein said endogenouspresenter protein is selected from the group consisting of: peptidylprolyl isomerases, Hsp90, steroid hormone receptors and cytoskeletalproteins.
 32. The method according to claim 31, wherein said endogenouspresenter protein is a peptidyl prolyl isomerase.
 33. A method forproducing a binary complex in a host, said method comprising:administering to said host an effective amount of a bifunctionalmolecule comprising a drug moiety and a ligand for a presenter proteinendogenous to said host; whereby a binary complex comprising saidbifunctional molecule and presenter protein is produced that exhibitsenhanced specificity for a target of said drug moiety target as comparedto a free drug control.
 34. The method according to claim 33, whereinsaid ligand for a presenter protein is a peptidyl prolyl isomerase. 35.A method for enhancing the selectivity of a drug for a target in a firstcell as compared to a second cell, said method comprising: contactingsaid first and second cells with a bifunctional molecule comprising saiddrug and a ligand for a presenter protein present in said second cellbut not in said first cell; whereby a binary complex comprising saidbifunctional molecule and presenter protein is produced in said secondcell but not said first cell.
 36. The method according to claim 35,wherein said drug moiety is an antimicrobial agent.
 37. The methodaccording to claim 35, wherein said ligand is a peptidyl prolylisomerase ligand.
 38. In a method of administering a drug to a host inneed of said drug, the improvement comprising: administering to saidhost an effective amount of a bifunctional molecule of less than about5000 daltons consisting of said drug or a fragment thereof covalentlylinked, either directly or through an optional linking group, to aligand for a presenter protein endogenous to said host.
 39. The methodaccording to claim 38, wherein said host is a mammalian host.
 40. Themethod according to claim 39, wherein said mammalian host is human. 41.The method according to claim 38, wherein said drug is a small molecule.42. The method according to claim 38, wherein said drug binds to anextracellular target.
 43. The method according to claim 38, wherein saiddrug binds to an intracellular target.
 44. The method according to claim43, wherein said presenter protein ligand is a peptidyl prolylisomerase.
 45. A method of making a bifunctional molecule comprising adrug moiety that exhibits at least one of enhanced affinity, specificityor selectivity as compared to the corresponding free drug, said methodcomprising: identifying a drug moiety; preparing a library ofbifunctional molecules comprising said drug moiety and a ligand for apresenter protein, wherein each bifunctional molecule shares a commonligand and drug moiety separated by a variable linking; and screeningsaid library to identify those members having at least one of enhancedaffinity, specificity or selectivity as compared to the correspondingfree drug.
 46. A pharmaceutical preparation comprising a bifunctionalmolecule according to claim
 1. 47. A kit comprising the pharmaceuticalpreparation according to claim 46 and instructions for use in atherapeutic method.